iv spanish portuguese biophysical congress · iv spanish portuguese biophysical congress program...

Book of Abstracts

IV Spanish Portuguese Biophysical

Congress

Zaragoza, SpainJuly 7-10, 2010

III

IVSPanISh PortugueSeBIophysIcalcongress

Table of contents

Program .........................................................................................................V

Committees ..................................................................................................IX

Plenary Lectures ........................................................................................... 1

Bruker and SBe awards................................................................................. 9

Invited Speakers ......................................................................................... 15

Short oral Presentations ............................................................................ 51

Posters ......................................................................................................... 89

Companies .................................................................................................269

List of Contributors in alphabetical order ...............................................273

List of Participants in alphabetical order ................................................295

V


Program

7th of July (Wednesday)

Afternoon session

16:00-18:30 Registration

18:30-19:00 Congress Opening

19:00-20:00

Opening Lecture:Joseprizo(U. Texas, USA)

20:00-21:00 Welcome reception

8th of July (Thursday)

Morning session

SIMPOSIUM I SIMPOSIUM II

Protein stability, folding and dynamicschairs:cláudioMgomes(ITQB, UNL, Oeiras)MargaritaMenéndez (IQFR, CSIC, Madrid)

Membrane biophysicschairs:MariaJoãoMoreno (FCTUC, Coimbra)Jesúspérez-gil (UCM, Madrid)

09:00-09:30 TeresapinheiroDep. Biol. Sciences. U. Warwick (UK)

sergigarcía-ManyesU. Columbia (USA)

09:30-10:00 VíctorMuñozCIB, Madrid

MargaridaBastosFCUP, Oporto

10:00-10:30 J.M.sánchezruizDep. Química-Física. U. Granada

FranciscoMonroyUCM, Madrid

10:30-11:00 Coffee break

11:00-11:15 Hugo Botelho Beatriz Apellaniz

11:15-11:30 Fernando Diez-García Ana P. Martins

11:30-11:45 Ana Cristina Sotomayor Pérez Oliveira Costa

12:00-12:45 Plenary lecture: MartaBruix (IQFR-CSIC, Madrid)

VI

12:50 Assembly of the SBE Board

Afternoon session

SIMPOSIUM III SIMPOSIUM IV

Biophysical analysis of proteins and macromolecular assemblieschairs:MaríaJoãoromão (FCT-UNL)o.llorca (CIB, Madrid)

Bioenergeticschairs:andreiaFernandes(IBMC, Porto)MilagrosMedina(U. Zaragoza, Zaragoza)

14:30-15:00 luislouraU. Coimbra

InêscardosopereiraITQB-UNL, Oeiras

15:00-15:30 JoséruizcastónCNB-CSIC

eduardorialCIB, Madrid

15:30-16:00 guillermoMontoyaCNIO, Madrid

MiguelangeldelarosaU. Sevilla

16:00-17:30 Coffee break & poster session

17:30-17:45 Inmaculada Pérez-Dorado Patricia Ferreira Neila

17:45-18:00 M. Angeles Jiménez. Paulo Gameiro

18:00-18:15 Esther Ortega Portero Jesus I Martinez

18:30-18:50 Plenary lecture: SBE Award (pauBernadó)

18:50-19:10 Plenary lecture: Bruker Award (Modestoorozco)

19:10-19:30 Plenary lecture: Bruker Award (Joséluisrodríguezarrondo)

19:30 SBE general assembly/SPBf general assembly

9th of July (Friday)

Morning session

SIMPOSIUM V SIMPOSIUM VI

advances in biophysical experimental methodschairs:Josérino (IMM - Fac. Medicina U. Lisboa)germánrivas (CIB. CSIC, Madrid)

Ion channel and transporterschairs:antonioFerrer-Montiel (UMH, Elche)graçasoveral (FF, U. Lisboa)

09:00-09:30 MariselaVélezI. Catálisis, CSIC, Madrid

TeresagiráldezU.Inves., Hospital Ntra Sra Candelaria(Tenerife)

09:30-10:00 anagarcía-sáezBiophysic/BIOTEC; Dresden (Germany)

FátimaMartelU. Oporto

10:00-10:30 MarcelonöllmanCBS CNRS-MontPellier (France)

VicenteaguilellaU. Jaime I, Castellón

VII



11:00-11:15 Adriana Martin de Aguilera M. P. Lostao

11:15-11:30 Sebastian Raja Joanna Bielanska

11:30-11:45 Rocio Esquembre Tomé Barbara Tavares

12:00-12:45 Plenary lecture:MiguelTeixeira(ITBQ, Universidade Nova de Lisboa)

Afternoon session

SIMPOSIUM VII SIMPOSIUM VIII

Biological Physicschairs:ruiDilão (IST, U. Técnica de Lisboa)Félixritort (U. Barcelona)

Cell Biophysicschairs:Jorgecarneiro(I. Gulbenkian de Ciência, Oeiras)luciaTabares (U. de Sevilla)

14:30-15:00 Jordigarcía-ojalvoUPC, Barcelona

XavierTrepatIBC-FM, Universitat de Barcelona

15:00-15:30 leonorcruzeiroU. Algarve, Faro

FilipaalvesI. Gulbenkian de Ciência, Oeiras

15:30-16:00 DanielnavajasU. Barcelona

Mariagarcía-parajoIBEC, Barcelona

16:00-17:30 Coffee break & poster session

17:30-17:45 Armindo Salvador Jaume Casademunt

17:45-18:00 Marta Ibañes Margarita Segovia Roldan

18:00-18:15 Alessandro Fiasconaro Rocio Ruiz Laza

18:30-19:30 Plenary lecture-Comité IUPAB: carlosBustamante(U. California, Berkeley, USA)

21:00 Congress dinner

10th of July (Saturday)

Morning session

SIMPOSIUM IX SIMPOSIUM X

single-moleculebiophysicsandnano-technologychairs:yannastier(ITQB, U. Nova de Lisboa)ricardoIbarra (INA, U. Zaragoza)

advances in biophysical computation and modellingchairs:Modestoorozco (IRB, Barcelona)armindosalvador (CNC, U. Coimbra)

09:00-09:30 christopheVieuLAAS-CNRS, Toulouse (France)

VictorguallarBSC, Barcelona

VIII

09:30-10:00 JoséIgnaciopascualBerlín (Germany)

XavierDauraICREA-UAB, Barcelona

10:00-10:30 Marianocarrión-VázquezI. Cajal-CSIC, Madrid

pauloMartelFST. U Algarve, Faro


11:00-11:15 Anabel Lostao José García de la Torre

11:15-11:30 Lara H Moleiro Federico Morán

11:30-11:45 Cristiano Bizarro Inma Rodriguez Cantalapiedra

12:00-13:00 Closing lecture: chrisM.Dobson (U. Cambridge, UK)

Closing

IX


Committees

Chairman Javier Sancho (BIFI, U. Zaragoza, Spain)

organizing CommitteeAlicia Alonso (U. País Vasco, Bilbao)Pierpaolo Bruscolini (BIFI, Zaragoza)Fernando Falo (BIFI, U. Zaragoza)Andreia S. Fernandes (IBMC, Porto)Carlos Gómez-Moreno (INA, U. Zaragoza)Milagros Medina (BIFI, U. Zaragoza)Manuela M. Pereira (ITQB, Oeiras)Jesús Pérez Gil (U. Complutense, Madrid)Germán Rivas (CIB-CSIC, Madrid)Javier Sancho (BIFI, U. Zaragoza)Nuno C. Santos (IMM, Lisboa)Adrián Velázquez-Campoy (BIFI, Zaragoza)

Scientific CommitteeAlicia Alonso (EHU, Bilbao)Peter Eaton (FC, Porto)Juan Carmelo Gómez-Fernandez (U. Murcia)Antonio Ferrer Montiel (UMH, Elche)Manuela M. Pereira (ITQB, Oeiras)Manuel Prieto (U. Tecnica Lisboa, Lisboa)Nuno C. Santos (U. Lisboa)Javier Sancho (U. Zaragoza)Cláudio M. Soares (ITQB, Oeiras)Lucía Tabares (U. Sevilla)

Conference SecretaryBeatriz AntolíIsabel Vidal


Plenary Lectures

3


plenarylectures

1/new Insights from old proteins: Structure, Folding, Stability and Interactions of ribonucleases Studied by nMr ................................................ 4Marta Bruix

2/the folding cooperativity of a protein is controlled by the topology of its polypeptide chain ............................................................................................... 5Carlos Bustamante, Elizabeth Shank, Ciro Cecconi, Jesse Dill and Susan Marqusee

3/Life on the edge: the generic nature of Protein Misfolding Disorders ....... 6Christopher M. Dobson

4/Structural Insights into the Mechanisms of neurotransmitter release and Information Processing in the Brain .................................................................. 7Cong Ma, Wei Li, Yibin Xu, Lijing Su, Josep Rizo

5/reductive elimination of reactive oxygen Species: Structural and Functional Insights .............................................................................................. 8Miguel Teixeira

4

plenarylectures 1

new Insights from old proteins: Structure, Folding, Stability and Interactions of ribonucleases Studied by nMrMarta Bruix1

1Instituto de Química Física Rocasolano, Serrano 119, 28006 Madrid, Spain.

Ribonucleases are interesting proteins and besides sharing a common ribonucleolytic activity, RNases play a number of different and crucial biological functions. Due to their small size and high stability, RNases have been extensively characterised and widely used as models in many biophysical studies. We have long employed NMR to determine the 3D structure, stability, folding, electrostatics, internal dynamics and intermolecular interactions of RNases, and used these data to gain insights into biological activities. Recently, modern recombinant expression methods have spurred the ready production of labelled human RNases opening the possibility to their study by NMR. Here, I will present our recent NMR results on human RNase 1 (HP-RNase) [1] and RNase 3 (ECP) [2,3] and also on bovine pancreatic RNase A [4,5]. On the bases of these data, the structure-activity relationship for RNase 1 will be described with especial emphasis on the structural factors affecting its binding with the specific RNase inhibitor (hcRI) [1]. RNase 3 is an Arg- and Pro- rich cytotoxic RNase implicated in asthma and the immune defence against parasites. Our results have shed light onto the origin of its extremely high conformational stability [2], and the interactions with membranes and hepa-rin mimetics [3]. Our results show that ECP binds to membrane and heparin simultaneously suggesting pathways for an efficient membrane internationalization. Also, we have shown that the N-terminal fragment (ECP 1-45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP 3D structure nor its high conformational stability seems to be required for protein cytotoxicity. Finally, using novel NMR pulse sequences [6] as well as conventional 2D and 3D spectra, the residual structure in the denaturated state of RNase A provoked by 40% acetic acid could be determined [5]. These solvent conditions promote the formation of 3D domain-swapping oligomers with additional biological and enzymatic activities. Our findings emphasise the role of the refolding intermediate with non-native X-Pro peptide bonds in oligomerisation and have generated implications for the mechanism of amyloidogenesis via 3D domain-swapping.

[1] Kover, K., Bruix, M., Santoro, J., Batta, G., Laurents, D.V., and Rico, M. (2008) J. Mol. Biol. 379, 953-965.[2] Laurents, D.V., Bruix, M., Jiménez, M.A., Santoro, J., Boix, E., Moussaoui, M., Nogués, M.V., and Rico. (2009) Biopolymers 91, 1018-1028.[3] García-Mayoral, M.F., Moussaoui, M., de la Torre, B.G., Andreu, D., Boix, E., Nogués, M.V., Rico, M., Laurents, D.V., and Bruix, M. (2010) Biophys. J. In press.[4] Bruix, M., Ribó, M., Benito, A., Laurents, D.V., Rico, M., and Vilanova, M. (2008) Biophys. J. 94, 2297-2305.[5] López-Alonso, J.P., Bruix, M., Font, J., Ribó, M., Vilanova, M., Jiménez, M.A., Santoro, J., González, C., and Laurents, D.V. (2010) J. Am. Chem. Soc. 132, 1621-1630.[6] Pantoja-Uceda, D., and Santoro, J. (2008) J. Magn. Reson. 195, 187-95.

5


plenarylectures 2

the folding cooperativity of a protein is controlled by the topology of its polypeptide chainBy Carlos Bustamante, Elizabeth Shank, Ciro Cecconi, Jesse Dill and Susan Marqusee

University of California, Berkeley, CA, USA

Proteins are complex functional molecules that tend to segregate into structural regions. Throughout evolution, biology has harnessed this modularity to carry out specialized roles and regulate higher-order functions such as allostery. Cooperative communication between such protein regions is important for catalysis, regulation, and efficient folding; indeed, lack of domain coupling has been implicated in the formation of fibrils and other misfolding patholo-gies. How domains communicate and contribute to a protein’s energetics and folding, howe-ver, is still poorly understood. Bulk methods rely on a simultaneous and global perturbation of the system (temperature or chemical denaturants) and can miss potential intermediates, thereby overestimating protein cooperativity and domain coupling. I will show that by using optical tweezers it is possible to mechanically induce the selective unfolding of particular regions of single T4 lysozyme molecules and establish the response of regions not directly affected by the force. In particular, I will discuss how the coupling between distinct domains in the protein depends on the topological organization of the polypeptide chain. To reveal the status of protein regions not directly subjected to force, we determined the free energy changes during mechanical unfolding using Crooks’ Fluctuation Theorem. We evaluate the cooperativity between domains by determining the unfolding energy of topological variants pulled along different directions. We show that topology of the polypeptide chain critically determines the folding cooperativity between domains and, thus, what parts of the folding/unfolding landscape are explored. We speculate that proteins may have evolved to select certain topologies that increase coupling between regions to avoid areas of the landscape that lead to kinetic trapping and misfolding.

6

plenarylectures 3

Life on the edge: the generic nature of Protein Misfolding Disorders Christopher M. Dobson

University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge CB2 1EW, UK

Natural proteins are a highly select group of molecules, and their properties have a number of very special characteristics when compared to random sequences of amino acids, one of which is the ability to fold to unique and often highly intricate structures, a characteristic that has enabled biological systems to generate a vast range of functions and an astonishing degree of specificity in their chemical processes [C.M. Dobson, Nature 426, 884-890 (2003)]. Because proteins are involved in every chemical process taking place within living systems, however, the failure of proteins to fold, or to remain correctly folded, can give rise to serious cellular malfunctions that frequently lead to disease [C.M Dobson, Trends Biochem. Soc. 24, 329-332 (1999)]. One particularly important group of such diseases is associated with the aggregation of misfolded proteins into remarkable thread-like structures known as amyloid fibrils, and includes disorders ranging from Alzheimer’s disease to late-onset diabetes, con-ditions that are becoming increasingly common in our aging populations. The manner in which the normal soluble forms of peptides and proteins can convert into these pathogenic amyloid structures is being uncovered by a wide variety of in vitro experimental studies along with theoretical simulations and bioinformatics studies [C.M. Dobson and F. Chiti, Annu. Rev. Biochem. 75, 333-366 (2006)]. As with folding, these studies are increasingly being linked to events occurring in vivo using a variety of strategies. Of particular interest are experiments designed to link the generic principles of misfolding and aggregation to the effects of such processes in model organisms such as Drosophila (the fruit fly) [Luheshi et al., PLoS. 5, e290 (2007)]. This talk will try to draw together some of the ideas that are emerging from recent work in our laboratory including evidence for the extremely narrow boundary between nor-mal and aberrant behaviour [Tartaglia et al., Trends Biochem. Soc. 32, 204-206 (2007)], and for the molecular mechanism of amyloid formation and proliferation [Knowles et al., Science 326, 1533-1537 (2009)]. Such studies are leading to an increasing level of understanding of the molecular origins and potential means of prevention of many of the diseases associated with misfolding including those that give rise to neurodegeneration.

http://www-dobson.ch.cam.ac.uk/Home.html

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plenarylectures 4

Structural Insights into the Mechanisms of neurotransmitter release and Information Processing in the BrainCong Ma, Wei Li, Yibin Xu, Lijing Su, Josep Rizo

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is a key event in interneuronal communication, and regulation of the release efficiency during distinct pro-cesses of presynaptic plasticity underlies multiple forms of information processing in the brain. Release is governed by a complex protein machinery that includes components with homologues in all types of intracellular membrane fusion, such as the SM protein Munc18-1 and the SNARE proteins synaptobrevin, syntaxin-1 and SNAP-25. In addition, neurotransmit-ter release depends on diverse proteins such as Munc13, synaptotagmin-1 and complexin, which have specialized functions that confer the exquisitely tight regulation of this process. Studies of the three-dimensional structures and interactions of these proteins are yielding key insights into the mechanisms of neurotransmitter release and its regulation. The three SNA-REs form a tight four-helix bundle called the SNARE complex that brings the synaptic vesi-cle and plasma membranes together and is key for membrane fusion. Munc18-1 also forms part of the core of the membrane fusion machinery, interacting with the SNAREs in different modes. Synaptotagmin-1 acts as a Ca2+ sensor in release by binding Ca2+ through its two C2 domains, which modulates interactions with the membranes and the SNARE complexes. Complexin binds tightly to the SNARE complex and plays dual active and inhibitory roles, in a tight interplay with synaptotagmin-1.

A key step for the regulation of neurotransmitter release is a structural transition of syn-taxin-1 whereby a so called-closed conformation of syntaxin-1 is opened to allow assembly of the SNARE complex. Munc18-1 binds to closed syntaxin-1, preventing SNARE complex formation, and also acts in downstream events that lead to membrane fusion. Munc13 is known to play an essential role in release through its MUN domain, and to mediate diver-se presynaptic plasticity processes through other domains, but the underlying mechanisms are unknown. Using NMR spectroscopy, we have now found that the Munc13 MUN domain dramatically accelerates the transition from the closed syntaxin-1-Munc18 complex to the SNARE complex, resulting in the formation of a 200 kDa Munc18-Munc13-SNARE macromo-lecular assembly. Additional NMR experiments show that Munc13 binds with moderate affi-nity to Munc18-SNARE complex assemblies, and more weakly to the syntaxin-1 SNARE motif, to the SNARE complex and to Munc18. These results suggest that Munc13 opens syntaxin through a catalytic mechanism involving cooperation between multiple weak interactions of the MUN domain with Munc18 and the SNAREs. These results support a model whereby ope-ning of syntaxin-1 by the Munc13 MUN domain is a crucial step in neurotransmitter release, and regulation of MUN domain activity by other domains of Munc13, as well as by other proteins involved in presynaptic plasticity, is fundamental for multiple forms of information processing in the brain.

8

plenarylectures 5

reductive elimination of reactive oxygen Species: Structural and Functional InsightsMiguel Teixeira

Instituto de Tecnologia Química e Biológica, Av. da República, 2780-157 Oeiras, Portugal

Living cells are, even if only transiently, exposed to dioxygen, with the resulting production of Reactive Oxygen Species (ROS), namely the superoxide anion and hydrogen peroxide. To counteract the deleterious effects of ROS, organisms evolved specialized enzymatic systems, mainly based on disproportionation mechanisms: superoxide dismutases (dismutation of the superoxide anion) and catalases (dismutation of hydrogen peroxide). However, these systems are not universal, and in particular in anaerobic bacteria and archaea, as well as in a few eukaryotic protozoa, completely different systems have been discovered, based on the reduc-tive elimination of ROS. One of these systems are the superoxide reductases, spread among anaerobic and facultative microorganisms, from the three life kingdoms. These enzymes share the same unique catalytic site, an iron-ion bound to four histidines and a cysteine, in a domain organized in a seven-stranded (3+4 sheet stranded) β-barrel that adopts an immunoglobulin-like fold. The reaction mechanism has been elucidated by a combination of pulse radiolysis, and electronic and Resonance Raman spectroscopies, of wild type and site directed mutants, and by determination of 3D crystallographic structures. In the reduced form, the SORs react with the superoxide anion with a diffusion-limited second order rate constant of ~109 M-1s-1, forming an iron-hydroperoxide species. Subsequently this transient may decay to an iron-hydroxide form or directly to the ferric resting form. The reduced form is rapidly re-formed by receiving electrons from rubredoxins, with a rate constant faster than 107 M-1s-1. Ano-ther system is exemplified by desulforubrerythrin (DRbr), a novel multidomain metalloprotein from Campylobacter jejuni. Each monomer is built by three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix-bundle domain harboring a µ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV-Visible, EPR and Resonance Ra-man spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 µmol H2O2 min-1mg-1. The 3D crystallographic structure was solved to 2Å resolution and reveals a tetrameric quaternary structure, with a high degree of domain swapping. This protein is named desulforubrerythrin (DRbr) and represents a novel example of the large diversity of the organization of domains exhibited by the rubrerythrin family.


Bruker and SBE awards

10

awards

SBE Award (Pau Bernadó)Bruker Award (Modesto Orozco)Bruker Award (José Luis Rodríguez Arrondo)

11


awards

Small-angle X-ray Scattering to Study Polydisperse Biomolecular SystemsPau Bernadó

Institute for Research in Biomedicine. [email protected]

Recent advances in synchrotron radiation sources and software modelling are the origin of the renaissance of SAXS to study biomolecular systems such as proteins and nucleic acids. SAXS is biophysical technique which is sensitive to the size, shape and weight properties of macromolecules in solution. Most of the available SAXS analysis methods for the structural characterization of biomolecules rely on the presence of a single species in solution. However, there are several biologically relevant scenarios where distinct species coexist and exchange with time. In this talk I will present some recent SAXS modelling strategies to address polydis-perse biological systems in a structurally comprehensive manner.

Highly flexible proteins, such as Intrinsically Disordered Proteins (IDPs), are extremely com-mon in Nature. These proteins can adopt very different sizes and shapes by sampling a vast conformational space. An ensemble optimization method (EOM) has been developed to study this family of proteins at structural level using SAXS [1]. EOM selects a set of conformations which simultaneously fit the SAXS profile.

SAXS can also be used to study self-oligomerization processes in proteins where different homomeric species coexist in solution. In these circumstances, the SAXS profile corresponds to the weighted average of the pure SAXS curves of all these species. The deconvolution of SAXS curves measured at different protein concentrations using chemiometrics approaches provides direct structural information of minor species as well as the thermodynamics gover-ning the process [2].

[1] Bernadó P, Mylonas E, Petoukhov MV, Blackledge M, Svergun DI. J.Am.Chem.Soc. 2007 129:5656-64.[2] Blobel J, Bernadó P, Svergun DI, Tauler R, Pons M. J.Am.Chem.Soc. 2009 131:4378-86.

12

awards

Biomolecular simulation: an overviewModesto Orozco

IRB, Barcelona

Molecular simulation is providing a new insight into biochemical system. The computer is becoming an ultramicroscope able to follow not only the nuclei flux, but also the electron movements implicit in any biological process. During my talk i will give my personal view on the state of the art in the field of biomolecular simulation, giving special emphassis on the field of applicability of these techniques

13


awards

especroscopia de infrarrojo de biomoléculasJosé Luis R. Arrondo

La espectroscopia de infrarrojo permite el estudio conformacional de diferentes moéculas biológicas. Es especialmente útil en el estudio de lípidos, proteínas y sistemas lipoproteicos, ya que no se ve afectada por la turbidez de la muestra y las bandas corespondientes a lípidos y proteínas no se solapan. Cuando empezamos nuestros estudios, se centraban principalmente en sistemas modelo de lípidos Se medía la posición de las bandas correspondientes a la fase apolar, la interfase y la fase polar.Posteriormente, utilizando tratamientos matemáticos, se realizó el estudio de proteínas descomponiendo la banda amida I. En una posterior aproximación al estudio de proteínas utilizamos el análisis propuesto por Noda (2DCOS) para obtener información de la interacción de bandas, principalmente en proteínas pero también en lípidos, en un proceso de perturbación de la molécula que estudiamos. Nuestra aprota-ción más reciente al estudio de proteínas por infrarrojo es lo que llamamos remoción de banda, donde combinamos el 2DCOS con la sustracción de bandas caraterísticas a fin de ver los cambios minoritarios que se producen en la proteína.


Invited Speakers

17

IVSpaniSh portugueSeBIophysIcalcongress

Invitedspeakers

1/role of ionizable residues in ion transport through bacterial porins ........ 20Vicente M. Aguilella, Antonio Alcaraz, M. Lidón López, Marcel Aguilella-Arzo, Elena García-Giménez, María Queralt-Martín.

2/Biophysical models for ion dynamics in cell polarization and apical growth ...................................................................................................... 21Filipa Alves

3/three-dimensional cryo-electron microscopy at subnanometer and quasi-atomic resolutions ............................................................................................. 22Daniel Luque, Josué Gómez, Roberto Marabini, José L. Carrascosa, José R. Castón

4/a Kinetic Mechanism for protein Folding in a Multi-funnel Free energy Landscape .......................................................................................................... 23Leonor Cruzeiro

5/effect of tyrosine nitration on the Structure-Function relations of Cytochrome c, a Bi-functional protein ............................................................. 24Miguel A. De la Rosa

6/nanomechanics of supported lipid bilayers: heads or tails? ...................... 25Sergi Garcia-Manyes

7/a feedback-free signaling switch in the notch-Delta pathway .................. 26David Sprinzak, Amit Lakhanpal, Lauren LeBon, Jordi Garcia-Ojalvo, Michael Elowitz

8/Structural movement of a gating ring in the BK channel ........................... 27Pablo Miranda, Diana Wesch, Jorge Contreras, Miguel Holmgren and Teresa Giraldez

9/peLe: protein energy Landscape exploration .............................................. 28Victor Guallar

10/Characterization of lipid/Dna and lipid/protein assemblies by fluorescence resonance energy transfer ......................................................... 30Luís Loura,, Catarina Madeira, Ana Coutinho,, Manuel Prieto

11/recent advances on the placental membrane transport of the water-soluble vitamins thiamine and folate .............................................................. 31Fátima Martel

18

12/Crystal structure of the mammalian cytosolic chaperonin CCt in complex with tubulin ....................................................................................................... 32Inés G. Muñoz, Hugo Yébenes, Min Zhou, Pablo Mesa,, Marina Serna, AhYoung Park, Elisabeth Bragado-Nilsson, Ana Beloso, Guillermo de Carcer, Marcos Malumbres, Carol V. Robinson, José M. Valpuesta & Guillermo Montoya

13/nanomechanics of living cells ..................................................................... 33Daniel Navajas

14/Mechanism of chromosomal segregation by Spoiiie ................................ 34Marcelo Nollmann

15/Scanning tunnelling spectroscopy on single molecules and molecular nanostructures .................................................................................................. 35Katharina J. Franke & Jose Ignacio Pascual

16/a new Membrane-Bound Complex in Bacterial anaerobic respiration .. 36Sofia S. Venceslau, Rita R. Lino, Inês A. C. Pereira

17/oxidative stress, thermogenesis and the evolution of the uncoupling proteins .............................................................................................................. 37Eduardo Rial, Jesús Jiménez-Jiménez, M. Mar González-Barroso.

18/the physical forces behind collective cell migration ................................ 38Xavier Trepat,

19/assembling a biological nanomotor on a nano-engineered surface ....... 39C. Vieu, J. Chalmeau, C. Thibault

20/addressing functionally relevant large-scale motions in proteins by molecular-dynamics simulation: the artJ case. .............................................. 40Roman Affentranger and Xavier Daura,

21/Biophysical characterization of antimicrobial peptides interaction with model membranes and biological implications on the mechanism of action 41Margarida Bastos

22/Cellulosome nanomechanics: moving from single cohesin modules to the whole complex................................................................................................... 42Mariano Carrión-Vázquez

23/an optical view of cell membrane compartmentalisation and functional implications ....................................................................................................... 43Maria F. Garcia-Parajo,

19


24/Quantification of protein-protein interactions within membranes by fluorescence correlation spectroscopy ............................................................ 44Ana J. García-Sáez, Jonas Ries, Mar Orzáez, Enrique Pérez-Payá3, Petra Schwille.

25/Studying protein-ligand interactions in a flexible enzyme ...................... 45P. Martel, V. Ribeiro, V. Teixeira

26/Continuous membrane mechanics: where physics meets cell biology ... 46Francisco Monroy

27/experimental Studies of primordial protein Folding ................................ 47Victor Muñoz

28/prion Folding and the Species Barrier ........................................................ 48Teresa J.T. Pinheiro

29/evolution and engineering of proteins ...................................................... 49Jose M. Sanchez-Ruiz

30/Looking at dynamic biofunctional surfaces at the nanoscale: initial stages of bacterial cell division .................................................................................... 50Marisel Velez

20

Invitedspeakers 1

role of ionizable residues in ion transport through bacterial porinsVicente M. Aguilella, Antonio Alcaraz, M. Lidón López, Marcel Aguilella-Arzo, Elena García-Giménez, María Queralt-Martín.

Laboratory of Molecular Biophysics. Department of Physics. University Jaume I. 12003 Caste-llón (Spain)

The large, highly conductive channels from the bacterial outer membrane known as porins [1] perform an important function of regulating the influx of nutrients and the extrusion of waste products. Still, its selectivity to charged solutes like small inorganic ions and some an-tibiotics may have some physiological relevance too. The bacterial porin OmpF from E. coli is here used as a model system for many other “large” channels whose transport properties are regulated by electrostatic interactions. Taking advantage that the atomic crystal structure of OmpF is known with good resolution, we combine electrophysiological experiments (reversal potential and single channel conductance measurements) with continuum electrostatic cal-culations (effective pKa’s of the protein ionizable residues and protein electric field sensed by the permeant ions across the pore) to gain useful insight on the role of both ion accessible and buried charged residues on the selective transport properties of the channel.

We show that selectivity measurements can also be helpful in providing information about the tertiary structure of protein channels whose atomic structure has not been resolved by X-ray or NMR methods, as is the case of the Meningococcal class 1 porin [2]. In cases where the crystal structure is known –as happens in OmpF, VDAC, alpha-hemolysin, and others– re-versal potential measurements give insights on the pKa of the residues involved in channel ion selectivity. Interestingly, by confronting OmpF selectivity measurements with a detailed analysis of the distribution of the protein charged residues, it is found that the channel se-lectivity is not completely ruled by the charges at the channel constriction (what is usually known as selectivity filter in many channels) but is the result of the concerted action of many other residues [3]. This conclusion is also supported by experiments with OmpF mutants in which key acidic residues of the constriction were replaced by neutral ones. The inversion of channel selectivity found in experiments with salts of multivalent cations [4] raises interesting questions about the current theories of charge inversion in charged interfaces [5].

[1] Nikaido, H. (2003) Microbiol. Mol. Biol. Rev. 67, 593-656.[2] Cervera, J., Komarov, A.G., and Aguilella, V.M. (2008) Biophys. J. 94, 1194-1202.[3] Alcaraz A., Nestorovich, E.M., Aguilella-Arzo, M., Aguilella, V.M., and Bezrukov, S.M. (2004) Biophys. J. 87, 943-957.[4] Alcaraz, A. Nestorovich, E.M., López, M.L., García-Giménez, E., Bezrukov S.M., and Aguilella, V.M. (2009) Biophys. J. 96, 56-66.[5] García-Giménez, E., Alcaraz, A., and Aguilella, V.M. (2010) Phys. Rev. E 021912, 1-7.

contactauthor:VicenteM.aguIlellaLaboratory of Molecular Biophysics. Department of Physics. University Jaume I. 12003 Castellón (Spain

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Invitedspeakers 2

Biophysical models for ion dynamics in cell polarization and apical growthFilipa Alves

Instituto Gulbenkian de Ciência, Oeiras, Portugal

Neurons, hyphae, root hairs and pollen tubes all share comparable mechanisms to regulate cell polarization, apical growth and chemotactic spatial orientation. In this work we are using the germinating pollen as a model system, as different experimental approaches have already produced high quality data, ranging from molecular biology to electrophysiology, imaging and transcriptomics. In these cells, ion dynamics plays a central role in the establishment of polarity and in the regulation of growth and chemotaxis. Experimentally, we observe and quantify complex temporal and spatial patterns of the ion fluxes across the plasma membrane and the free ion concentrations in the cytoplasm. Based on the available data, we are develo-ping computational models integrating ion fluxes and transporters distribution. These models predict the observed intracellular ion gradients, shedding light on the minimal necessary conditions to establish these gradients and providing a simple rationale for species-specific differences.

contactauthor:FilipaalVesInstituto Gulbenkian de Ciência, Oeiras, Portugal

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Invitedspeakers 3

three-dimensional cryo-electron microscopy at subnanometer and quasi-atomic resolutionsDaniel Luque1, Josué Gómez1, Roberto Marabini1, José L. Carrascosa1, José R. Castón1

1Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología. Conse-jo Superior de Investigaciones Científicas. C/ Darwin nº 3, Cantoblanco. 28049 Madrid. Spain.

Many macromolecular assemblies or biological nanomachines, including virions, have elu-ded to be studied by X-ray crystallography and/or NMR because they are too large or too heterogeneous. Three-dimensional structures of these macromolecular complexes have, ne-vertheless, been successfuly derived from cryo-electron microscopy (3D cryo-EM) analysis and single-particle reconstruction techniques, first up to subnanometer resolution, and more recently at near-atomic resolution. We have applied this methodology to three viral systems with different levels of complexity: infectious bursal disease virus (IBDV), Penicilium chryso-genum virus (PcV), and rabbit haemorragic disease virus (RHDV).

The 3D structure of IBDV subviral particles to 5.0 Å shows not only secondary-structure elements (SSE) corresponding to a helices and b sheets, but also the densities for loops, The intrinsic flexibility of the C-terminal region of the capsid protein VP2 is also revealed after comparison with its atomic resolution structure. Finally, some voluminous side chains are defined in specific a helices.

PcV structure has been solved at 8.0 Å resolution. The structural asymmetric unit boundaries were established based on its compactness and contacts with neighboring densities. The capsid protein has a high content in rod-like densities characteristic of a helices, forming a re-peated a-helical core indicative of gene duplication. The spatial arrangement of the a-helical core resembles that found in the L-A virus capsid protein, indicating a conserved basic fold, and could provide insight into the structural evolutionary relationships between dsRNA viru-ses. Due to its numerous interactions with the inner surface of the capsid, the encapsidated genome conforms the icosahedral symmetry and is partially visualized as tubular densities corresponding to an A-form duplex.

The resolution of empty RHDV particles was estimated to be 9.0 Å. Considering that all cali-civirus coat proteins make a similar T = 3 capsid consisting of 90 dimers, and the relatively high similarity in amino acid sequence exhibited among these capsid proteins, we propose a model for its capsid proteín that fits properly in the T = 3 capsid structure. Our cryo-EM-validated pseudo-atomic model shed light into the mechanism that allows RHDV coat protein to switch among quasi-equivalent conformational states to achieve the appropriate curvature for the formation of a closed shell. The VP60 N-terminal arms are visualized forming extensi-ve domain-swapping interactions in their dimeric partners.

contactauthor:Josér.castónDepartamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología Consejo Superior de Investigaciones Científicas C/ Darwin nº 3, Cantoblanco. 28049 Madrid. Spain

23


Invitedspeakers 4

a Kinetic Mechanism for protein Folding in a Multi-funnel Free energy LandscapeLeonor Cruzeiro1

1CCMAR and FCT, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.

According to the thermodynamics hypothesis [1] and the associated theory of a funnel-sha-ped free energy landscape [2-5], the native structure of proteins is that which minimizes its free energy. In this view, the protein folding problem is essentially solved [4,5] since finding a protein structure from its sequence, in a computer, depends solely on having a sufficiently accurate potential to describe the interactions of protein atoms with one another and on the availability of enough computer power to explore the protein conformational space. A ques-tion [6] that lurks underneath the funnel theory is whether the potential energy functions that are successful in the modelling of proteins and of their interaction with ligands do in-deed lead to a funnel-shaped free energy landscape. In this talk, evidence for a multi-funnel landscape [7,8] is presented and a new, detailed, kinetic, mechanism for protein folding is put forward that can explain both reliable folding in a multi-funnel free energy landscape and the occasional misfolding [7,8]. Finally, the talk ends with a discussion of how this new kinetic mechanism fits with the existing experimental data on protein folding and misfolding.

[1] Anfinsen, C.B. (1973) Science 181, 223–230.[2] Bryngelson, J.D., Onuchic, J.N., Socci N.D., and Wolynes, P.G. (1995) Proteins 21, 167-195. [3] Dill, K.A. and Chan, H.-S. (1997) Nature Struct. Biol 4, 10-19.[4] Wolynes, P.G. (2005) Quart. Revs. Biophys. 38, 405-410.[5] Service, R.F. (2008) Science 321, 784-786.[6] Cruzeiro-Hansson, L. and Silva, P.A.S. (2001) J. Biol. Phys. 27, S6-S9.[7] Cruzeiro, L. (2008) J. Phys. Org. Chem. 21, 549-554.[8] Cruzeiro, L. and Lopes, P.A. (2009) Mol. Phys. 107, 1485–1493.

contactauthor:leonorcruzeIroCCMAR and FCT, Universidade do Algarve Campus de Gambelas, 8005-139 Faro, Portugal

24

Invitedspeakers 5

effect of tyrosine nitration on the Structure-Function relations of Cytochrome c, a Bi-functional proteinMiguel A. De la Rosa

Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla-CSIC, Spain

Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Respiratory cytochrome c, with 4-6 tyrosine resi-dues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mito-chondrial respiratory electron transport chain and, on the other hand, it serves as a cytoplas-mic apoptosis-triggering agent.

First, we have analyzed the nitration-induced changes in secondary structure, thermal stabi-lity, heme environment, alkaline transition and molecular dynamics of the five monotyrosine mutants of human cytochrome c - which have all their tyrosine residues but one replaced by phenylalanines. The resulting data, along with the functional analyses of the mutants, suggest that the specific nitration of Tyr46 and Tyr48 - which are both close to the heme propionate groups - and that of the solvent-exposed Tyr74 impair the electron transfer to (horse) cyto-chrome c oxidase, enhance the peroxidase activity of cytochrome c and block its ability to activate caspase-9 [1, 2].

In addition, a comparative proteomic analysis with human, algal and plant cytochrome c – and cell extracts from the respective organisms – has allowed us to identify novel proteins that could act as physiological partners of cytochrome c under normal or programmed cell-death conditions. The finding of new protein partners of cytochrome c in differently evolved orga-nisms will help us to understand, in a global way, the function of non-nitrated and nitrated cytochrome c in cell metabolism.

This work has been supported by the Spanish Ministry of Science and Innovation (Grant BFU2009-07190).

[1] Rodríguez-Roldán, V., García-Heredia, J.M., Navarro, .JA., Hervás, M., De la Cerda, B., Molina-Heredia, F.P., De la Rosa, M.A. (2006) Biochem. Biophys. Res. Commun. 346, 1108-1113.[2] García-Heredia, J.M., Díaz-Moreno, I., Nieto, P.M., Orzáez, M., Kocanis, S., Teixeira, M., Pérez-Payá, E., Díaz-Quintana, A., De la Rosa, M.A. (2010) Biochim. Biophys. Acta – Bioenergetics (in press).

contactauthor:Miguela.DelarosaInstituto de Bioquímica Vegetal y Fotosíntesis Centro de Investigaciones Científicas Isla de la Cartuja Universidad de Sevilla-CSIC, Spain

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Invitedspeakers 6

nanomechanics of supported lipid bilayers: heads or tails?Sergi Garcia-Manyes

Department of Biological Sciences, Columbia University, New York, 10027, NY, USADepartament de Química Física, Universitat de Barcelona, 08028, Spain

Understanding the effect of mechanical stress on membranes is of primary importance in biophysics. Here we use force spectroscopy AFM to quantitatively characterize the nano-mechanical stability of supported lipid bilayers as a function of their chemical composition. The onset of plastic deformation reveals itself as a repetitive jump in the approaching force curve, which represents a molecular fingerprint for the bilayer mechanical stability. By sys-tematically probing a set of chemically distinct supported lipid bilayers, we first show that both the headgroup and tail have a decisive effect on their mechanical properties. While the mechanical stability of the probed SLBs linearly increases by 3.3 nN upon the introduction of each additional –CH2– in the chain, it exhibits a significant dependence on the phospholipid headgroup, ranging from 3 nN for DPPA to 66 nN for DPPG. Furthermore, we also quantify the reduction of the membrane mechanical stability as a function of the number of unsatura-tions and molecular branching in the chemical structure of the apolar tails. Finally, and con-trary to previous belief, we demonstrate that upon introduction of cholesterol and ergosterol, the mechanical stability of membranes not only increases linearly in the liquid phase (DLPC) but also for phospholipids present in the gel phase (DPPC). Our results are discussed in the framework of the continuum nucleation model. This work highlights the compelling effect of subtle variations in the chemical structure of phospholipid molecules on the membrane response when exposed to mechanical forces, a mechanism of common occurrence in nature.

contactauthor:sergigarcía-ManyesDepartment of Biological Sciences Columbia University, New York, 10027, NY, USA

26

Invitedspeakers 7

a feedback-free signaling switch in the notch-Delta pathwayDavid Sprinzak1, Amit Lakhanpal1, Lauren LeBon1, Jordi Garcia-Ojalvo2, Michael Elowitz1

1Howard Hughes Medical Institute, Division of Biology and Department of Applied Physics, California Institute of Technology, Pasadena, California, USA2Departament de Física i Enginyeria Nuclear, Universitat Politècnica de Catalunya, Terrassa, Spain

In this talk I will discuss the impact of cis-inhibition in the dynamics of the Notch-Delta cell-cell communication pathway. We show, by means of a combination of single-cell measure-ments and mathematical modeling, that intracellular mutual inhibition of the Notch and Delta proteins leads to an ultrasensitive switch that determines which cell sends a signal and which cell receives it, without the need of feedback mechanisms downstream of the communication channel [1]. The influence of this switch on pattern and boundary formation will be also discussed.

[1] Sprinzak, D., Lakhanpal, A., Lebon, L., Santat, L.A., Fontes, M.E., Anderson, G.A., Garcia-Ojalvo, J., Elowitz, M.B. (2010) Nature 465, 86-91.

contactauthor:JordigarcIa-oJalVoDepartament de Física i Enginyeria Nuclear Universitat Politècnica de Catalunya, Terrassa, Spain

27


Invitedspeakers 8

Structural movement of a gating ring in the BK channelPablo Miranda2, Diana Wesch2, Jorge Contreras1, Miguel Holmgren1 and Teresa Giraldez2

1National Institute of Neurological Diseases and Stroke, NIH, Bethesda, USA2Unidad de Investigacion, Hospital Univ. Ntra Sra de Candelaria, Sta Cruz de Tenerife, Spain

The calcium sensitivity of the large conductance voltage- and calcium-activated potassium channel (BK) that is modulated both by membrane voltage and by intracellular calcium con-centration resides in a large, cytosolic domain. Opening of the channel requires less energy when calcium binds to this region, but the molecular mechanism underlying this process is unclear. The structure of a prokaryotic, tetrameric, calcium activated, potassium channel (MthK), and very recently, the structure of the calcium activation domain of the human BK channel suggest that its intracellular calcium sensor domains (RCK) form a ring structure (called the gating ring). The gating ring would expand upon calcium binding, presumably applying a force on the channel gate to make its opening more energetically favorable. In this study we measure movements within the putative BK gating ring domain, using FRET. Using a transposition-based approach, we have labeled the RCK and calcium-binding domains with ei-ther CFP or YFP. Hetero-tetramers that contain both CFP and YFP tagged domains have been formed. We have used Patch-Clamp Fluorometry to obtain fluorescence spectra of excised oocyte membrane patches expressing YFP/CFP heterotetramers with insertions in different sites along the gating ring region, including the linker between RCK1 and RCK2 domains, the calcium bowl and RCK2. Depending on the site that is tagged, we detect changes in FRET due to changes in voltage (insertions in the RCK1-RCK2 linker) or in calcium concentration (insertion in RCK2). No changes are observed when the probe in inserted in the Calcium bowl. These results are consistent with the model where a “gating ring” expands in response to increased calcium concentrations.

Acknowledgements and funding: this work is supported by Consolider-Spanish Ion Channel Initiative CSD2008_00005 (Ministerio de Ciencia e Innovación –MICINN- Spain). DW is su-pported by a FPI fellowship (MICINN). TG is supported by the Miguel Servet Program (ISCIII, MICINN). We wish to thank Deepa Srikumar at the NIH/NINDS for technical support in this project.

contactauthor:teresagIralDezUnidad de Investigacion Hospital Univ. Ntra Sra de Candelaria Sta Cruz de Tenerife, Spain

28

Invitedspeakers 9

peLe: protein energy Landscape explorationVictor Guallar1

1ICREA Research Professor, Life Science Department, Barcelona Supercomputing Center, Jordi Girona, 29, 08034 Barcelona (Spain). Email: [email protected]

We have developed a novel computational methodology to perform protein energy landscape ex-ploration (PELE).[1] PELE constitutes a remarkable advance over conventional techniques to map protein and protein-ligand dynamics. This method, which combines protein structure prediction te-chniques with a metropolis algorithm, is capable of describing the all-atom migration pathway of a large ligand in approximately 100 hours of CPU. PELE has already been cited in three independent review articles, [2-4] and has been underlined as one of the most promising software to study pro-tein-ligand recognition mechanism. Using PELE, for example, we have obtained the ligand migra-tion mechanism in myoglobin, in cytochrome P450, in the fatty acid binding proteins[1], and the ligand deligation and migration mechanism in the Truncated Hemoglobin-II from Mycobacterium tuberculosis[5]. PELE’s heuristic algorithm is based on consecutive iteration of three main steps:

1) Local perturbation: includes alpha carbon displacement, following one of the lowest nor-mal modes, and ligand displacement (if a ligand is present)

2) Side chain sampling. The algorithm proceeds by placing all side chains (and ligand flexible groups) local to the atoms perturbed in the first step.

3) Minimization. The last step involves the minimization of a region including, at least, all residues local to the atoms involved in steps 1 and 2. The minimization is intended to generate a backbone response to the initial local per-turbation and to possible side chains rota-tion in the first steps.

These steps form a move which is accepted (a new minima) or rejected based on a Me-tropolis criterion for a given temperature. The collection of accepted steps forms a sto-chastic trajectory.

Our group will be developing a server, at the Barcelona Supercomputing Center, where we will offer PELE (free) for the academic re-searchers. In this talk we will present the capabilities of PELE and its recent applications to map protein and protein-ligand dynamics[6].

[1] K. W. Borrelli, A. Vitalis, R. Alcantara and V. Guallar, PELE: Protein energy landscape exploration. A novel Monte Carlo based technique, Journal of Chemical Theory and Computation. 1 (2005) 1304-1311.

[2] K. Pant and U. Dutta, Software packages for studying diffusion pathways of gases in proteins, BRL. 2 (2007) 273-286.[3] H. X. Lei and Y. Duan, Improved sampling methods for molecular simulation, Current Opinion In Structural Biology. 17 (2007) 187-191.[4] J. C. Schon and M. Jansen, Prediction, determination and validation of phase diagrams via the global study of energy landscapes, International Journal of Materials Research. 100 (2009) 135-152.[5] V. Guallar, C. Lu, K. Borrelli, T. Egawa and S.-R. Yeh, Ligand Migration in the Truncated Hemoglobin-II

Figure1. Sampling procedure in PELE

29


from Mycobacterium tuberculosis: The role of G8 tryptophan, J. Biol. Chem. 284 (2009) 3106-3116.[6] K. W. Borrelli, B. Cossins and V. Guallar, Exploring hierarchical refinement techniques for induced fit docking with protein and ligand flexibility, J. Comp. Chem. ASAP (2010)

contactauthor:VictorguallarICREA Research Professor, Life Science Department,Barcelona Supercomputing Center Jordi Girona, 29, 08034 Barcelona (Spain) Email: [email protected]

30

Invitedspeakers 10

Characterization of lipid/Dna and lipid/protein assemblies by fluorescence resonance energy transferLuís Loura1,2, Catarina Madeira3, Ana Coutinho4,5, Manuel Prieto4

1Faculdade de Farmácia, Universidade de Coimbra, Portugal2Centro de Química de Coimbra, Universidade de Coimbra, Portugal3Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engi-neering, Instituto Superior Técnico, Lisboa, Portugal4Centro de Química Física Molecular, Complexo I, Instituto Superior Técnico, and Institute of Nanosciences and Nanotechnology, Lisboa, Portugal5Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Portugal

Complexes between cationic liposomes and DNA (lipoplexes) are important nonviral-based vectors in gene therapy. Many efforts have been made to fully characterize lipoplex structure and its dependence on environmental variables, because it is the only way to understand, improve, and control their transfection efficiency. On the other hand, electrostatic interac-tions between negatively charged membranes and basic peptides/protein domains have been implicated as the driving force for several important processes, often involving membrane aggregation, fusion and/or phase separation, and have been recently implicated in amyloid-like fiber formation. Förster (Fluorescence) Resonance Energy Transfer (FRET) is a powerful spectroscopic technique for molecular characterization of both lipid/DNA and lipid/protein assemblies. The sensitivity of FRET for distances of 10 up to 100 Å is particularly useful to retrieve information on the relative distribution of these components in the distance range for which electrostatic interactions are most operative. Formalisms useful in the analysis of FRET data for relevant supramolecular arrangements are presented and illustrated by recent examples of both lipid/DNA [1-3] and lipid/protein(peptide) [4,5] studies.

[1] Madeira, C., Loura, L.M.S., Aires-Barros, M.R., Fedorov, A., Prieto, M. (2003) Biophys. J. 85, 3106-3119.[2] Madeira, C., Loura, L.M.S., Prieto, M., Fedorov, A., Aires-Barros, M.R. (2007) Eur. Biophys. J. 36, 609-620.[3] Madeira, C., Loura, L.M.S., Prieto, M., Fedorov, A., Aires-Barros, M.R. (2008) BMC Biotechnology, 8, 20.[4] Loura, L.M.S., Coutinho, A., Silva, A., Fedorov, A., and Prieto, M. (2006) J. Phys. Chem. B 110, 8130-8141.[5] Coutinho, A., Loura, L.M.S., Fedorov, A., and Prieto, M. (2008) Biophys. J., 95, 4726-4736.

contactauthor:luíslouraFaculdade de Farmácia Universidade de Coimbra, Portugal

31


Invitedspeakers 11

recent advances on the placental membrane transport of the water-soluble vitamins thiamine and folateFátima Martel1

1Department of Biochemistry, Faculty of Medicine of Porto, University of Porto, Portugal

Thiamine (vitamin B1), a member of the water-soluble vitamin family, is essential for normal cellular functions, growth, and development. In its coenzyme form, thiamine pyrophosphate, it plays a critical role in normal carbohydrate metabolism, by participating in the decarboxyla-tion of α-keto acids such as pyruvic and ketoglutamic acid, and in transketolation reactions. Folate (folic acid) is another essential micronutrient for normal cellular functions, growth, and development. The one-carbon derivatives of this water-soluble vitamin function as coenzymes necessary for the synthesis of purine and pyrimidine precursors of nucleic acids, the metabo-lism of certain amino acids, and the initiation of protein synthesis in mitochondria.

Thiamine and folate are particularly important during pregnancy, for normal growth and de-velopment of the fetus, being obtained from the maternal blood through placental transport. The strict importance of these vitamins to fetal and pregnancy health is the motor for the cu-rrent and emerging research on the molecular mechanisms of their placental cell membrane transport.

A review of some recent findings concerning the placental transport of folate and thiami-ne will be made. These findings permit to conclude that (1) placental folate and thiamine membrane transport are complex processes that seem to involve several different transport systems for each of these vitamins, suggesting that some “transport redundancy” is likely to contribute to an efficient transfer of the vitamins through the placenta, ensuring an adequate supply to the developing fetus even in cases of maternal marginal vitamin status, and (2) placental transport of these vitamins may be changed by several dietary substances, present in commonly consumed beverages, by therapeutic drugs, by drugs of abuse and in some pathological conditions (intrauterine growth restriction) and this may threaten the normal development and growth of the fetus.

This work was supported by Fundação para a Ciência e a Tecnologia (FCT) and Programa Ciên-cia, Tecnologia e Inovação do Quadro Comunitário de Apoio (PTDC/SAU-FCF/67805/2006).

contactauthor:FátimaMartelDepartment of Biochemistry, Faculty of Medicine of Porto University of Porto, Portugal

32

Invitedspeakers 12

Crystal structure of the mammalian cytosolic chaperonin CCt in complex with tubulin Inés G. Muñoz1, Hugo Yébenes2, Min Zhou3, Pablo Mesa1,2, Marina Serna2, AhYoung Park3, Elisabeth Bragado-Nilsson1, Ana Beloso2, Guillermo de Carcer4, Marcos Malumbres4, Carol V. Robinson3, José M. Valpuesta2 & Guillermo Montoya1

1Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fdez. Almagro 3, 28029 Madrid, Spain2Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Darwin, 3, 28049 Madrid, Spain.3Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Department of Chemistry, Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK.4Cell Division & Cancer Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fdez. Almagro 3, 28029 Madrid, Spain. E-mail: [email protected]

Protein folding in the cell is assisted by a large group of proteins termed molecular chape-rones1, one of the most important members being the chaperonins or Hsp60s (Heat Shock Proteins of 60 kDa). The eukaryotic cytosolic chaperonin CCT2 (chaperonin containing TCP-1, also known as TRiC) is the most complex of all chaperonins, a 1 MDa oligomer built by two identical rings, each composed of single copies of eight different 60kDa subunits called α, β, γ, ζ, ε, δ, θ and η. This macromolecular complex has crucial relevance in several essential biological processes, emerging as a key molecule due to its role in the folding of many important proteins including actin, α and β tubulins 3. An electron density map at 5 Å resolution has enabled us to build the CCT complex, revealing the presence of a substrate in the inner cavities of both octagonal rings. Here we present the crystal structure of this protein machine in complex with tubulin, providing information about the molecular mechanism by which this macromolecular complex aids the tubulin folding process. Our data provide the structural basis for understanding the function of this molecular machine.

contactauthor:guillermoMontoyaMacromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fdez. Almagro 3, 28029 Madrid, Spain E-mail: [email protected]

33


Invitedspeakers 13

nanomechanics of living cellsDaniel Navajas

Unitat de Biofisica i Bioenginyeria, Facultat de Medicina – Universitat de Barcelona and Insti-tut de Bioenginyeria de Catalunya. Barcelona. Spain.

Mechanical properties of the cell play a key role in many important cell func¬tions inclu-ding contraction, migration, proliferation and differentiation. Moreover, these processes are mediat¬ed by the ability of the cell to feel and respond to mechanical stresses and to mecha-nical features of the microenvi¬ronment. The cell mechanical behavior is increasingly recog-nized as a key de¬terminant of the normal cell function and of its alteration under patholo-gical conditions. However, the mechanisms underlying the mechanical properties of the cells and their interplay with the mechanical microenviroment remain largely unknown. Recent development of nanotechnologies specially suited for probing biological samples allows the study of mechanics at the single cell level. We developed a novel device to subject cultured cells to dynamic stretch [1]. Combination of this device with nanomanipulation of microbeads attached to the cell surface allowed us to reveal a universal law of strain-induced transient cell fluidization [2]. A novel technique to map traction forces exerted by cultured cells in a stretchable substrate allowed us to show that strain-induced fluidization is associated with a transient disruption of the actin-myosin machinery [3]. We have investigated the physical laws that govern cell dynamics by particle nanotracking, showing that cytoskeleton remode-ling is a thermically activated process mediated by ATP [4]. With atomic force microscopy (AFM) we have shown that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors [5]. AFM is a powerful tool for probing cell nanomechanics. However, contact area between the tip and the cell is not precisely defined in conventional AFM tips. Novel flat ended cylindrical tips [6] allowed us to reveal a diffe-rential mechanical response of lung epithelial cells to compressive and tensile local stresses. Neutrophil mechanics was measured with AFM in patients with advanced hypoxemic chronic obstructive pulmonary disease (COPD) before and after bilateral lung transplantation, and compared with measurements taken in healthy nonsmokers. Young’s modulus of neutrophils from patients with COPD was significantly greater than controls. Neutrophil stiffness decrea-sed after lung transplantation showing no significant differences with healthy nonsmokers. Neutrophil stiffening in COPD patients may be related to the abnormal inflammatory res-ponse of the lung. Neutrophil improvement after lung transplantation suggests decreased inflammatory pulmonary and systemic responses.

[1] Trepat X, Grabulosa M, Puig F, Maksym GN, Navajas D, Farre R. (2004) Am J Physiol Lung Cell Mol Physiol 287, L1025-L1034.[2] Trepat X, Deng L, An SS, Navajas D, Tschumperlin DJ, Gerthoffer WT, Butler JP, Fredberg JJ. (2007) Nature 447:592-595. [3] Gavara N, Roca-Cusachs P, Sunyer R, Farré R, Navajas D. (2008) Biophys J 95:464–471. [4] Sunyer R, Ritort F, Farré R, Navajas D. (2009) Phys Rev E 79:051920.[5] Sunyer R, Trepat X, Fredberg JJ, Farré R, Navajas D. (2009) Phys Biol 6: 025009.[6] Rico F, Roca-Cusachs P, Sunyer R, Farré R, Navajas D. (2007) J Mol Recognit 20 459-466.

contactauthor:DanielnaVaJasUnitat de Biofisica i Bioenginyeria, Facultat de Medicina Universitat de Barcelona and Institut de Bioenginyeria de Catalunya. Barcelona. Spain.

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Invitedspeakers 14

Mechanism of chromosomal segregation by SpoiiieMarcelo Nollmann

CNRS

ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stran-ded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transfer. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which ~3Mbp of chromosomal DNA are trans-ported across a division septum by the SpoIIIE ATPase. We show that SpoIIIE translocates DNA at ~5kbp/s while specifically interacting with highly skewed chromosomal sequences (SRS) that guide its directional motion. In addition, our data suggests that SpoIIIE assembles in a compartment-specific manner. By using both in vitro and in vivo biophysical methods, we address the mechanism of sequence recognition and complex assembly by SpoIIIE. First, we use single-molecule and in bulk fluorescence methods to tease out the kinetic steps involved in SRS recognition by SpoIIIE. Second, we use photo-activated localization microscopy, a re-cently developed super-resolution microscopy method, and Number & Brightness analysis to directly visualize the architecture and the assembly dynamics of the SpoIIIE complex in live cells.

contactauthor:MarcelonollMannCNRS

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Invitedspeakers 15

Scanning tunnelling spectroscopy on single molecules and molecular nanostructuresKatharina J. Franke & Jose Ignacio Pascual

Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, Berlin 14195, Germany

The fundamentals of molecular electronics lies in the use of the strongly nonlinear trans-port characteristics through single molecules to induce effects like rectification of an electric current or negative-differential resistance, crucial for the operation of logic devices. On the first grounds such non-linear effects are a consequence of the orbital structure of the mo-lecules, which become localized resonances so far the molecule is weakly interacting with the source/drain electrodes. Scanning tunneling microscope is an ideal tool to investigate electronic transport because it is able to explore elastic and inelastic scattering phenomena in the electronic transport through a single molecule and at the same time provide a detai-led characterization of the role of molecular orientation, conformation and contact with the substrate electrode.

In this presentation we will show scanning tunneling spectroscopy results on several mole-cular systems ranging from model fullerene cages to molecular conformational switches. In particular, we are interested in resolving the role of molecular orientation and conformation on their electronic and vibrational fingerprint. Molecular resonances alignment can be related to various parameters related to the surrounding medium, like the Polarizability of neighbor molecules, the distance to the metal surface, and/or the presence of local defects like metal steps. Besides on the specific alignment we find that molecular orbital splitting arises as a response to the local adsorption phenomenology.

We have also investigated the role of the metal surface in the spectroscopic electronic trans-port fingerprint by studying several aromatic molecular architectures with a three dimensio-nal structure. On noble metals these compounds are weakly absorbed, exposing their aro-matic moieties to the formation of non-covalent intermolecular bonds mediated by their π states. When fullerenes are added to the molecular films, we find that complex three dimen-sional supra-molecular structures are spontaneously formed, in which a fullerene cage is held by such π-active compounds and electronically decoupled from the metal surface. Weak coupling regime is particularly interesting for investigation molecular elementary excitations because their larger lifetime. Scanning tunneling spectroscopy resolves negative differential resistance in the electron transport spectrum through these supramolecules, which we attri-bute to the electronic decoupling of fullerene states from the metal surface. Our results fur-ther probe that three-dimensional "molecular clusters" build from a basic bottom-up approach are ideal systems to explore the rules of electronic transport though single molecules, also in the regime weak coupling with the metal electrodes

contactauthor:JoseIgnaciopascualInstitut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, Berlin 14195, Germanyl

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Invitedspeakers 16

a new Membrane-Bound Complex in Bacterial anaerobic respirationSofia S. Venceslau, Rita R. Lino, Inês A. C. Pereira

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal

Bacteria have very flexible and branched respiratory chains, reflecting their ability to adapt quickly to a changing environment. Bacterial respiratory complexes (BRC) usually have sim-pler compositions than their eukaryotic counterparts and are therefore useful models to un-derstand the molecular mechanisms involved in energy conservation. Furthermore, bacteria contain many unique BRC whose architecture has a highly modular character, with different arrangements of modules giving rise to different proteins and physiological functions.

Here we describe a new respiratory complex that was isolated as a major protein present in the membranes of Desulfovibrio vulgaris Hildenborough [1], a model sulfate reducing orga-nism. The complex, which was named Qrc, is the first representative of a new family of redox complexes. It has three subunits related to the complex iron-sulfur molybdoenzyme (CSIM) family [2], and a multiheme cytochrome c. It binds six hemes c, one [3Fe-4S]+1/0 cluster, several interacting [4Fe-4S]2+/1+ clusters, but no molybdenum. Qrc is related to the alter-native complex III [3], but has the reverse catalytic activity, acting as a Type I cytochrome c3:menaquinone oxidoreductase. The qrc genes are found in the genomes of deltaproteobac-terial sulfate reducers, which have periplasmic hydrogenases and formate dehydrogenases that lack a membrane subunit for reduction of the quinone pool. In these organisms, Qrc acts as a menaquinone reductase with electrons from periplasmic hydrogen or formate oxidation. Binding of a menaquinone analogue affects the EPR spectrum of the [3Fe-4S]+1/0 cluster, indicating the presence of a quinone-binding site close to the periplasmic subunits. This new complex is a striking evolutionary cross point in the CISM family, in which one function has been lost while another is evolving. Furthermore, Qrc is the first respiratory complex from sulfate reducers to have its physiological function clearly elucidated, which is an important contribution to our understanding of biological sulfate reduction, a process with high environ-mental and evolutionary significance.

[1] Venceslau S.S., Lino R. and Pereira I.A.C. (2010) J. Biol. Chem. published online May 24, 2010 as doi:10.1074/jbc.M110.124305[2] Rothery, R. A., Workun, G. J., and Weiner, J. H. (2008) Biochim Biophys Acta-Biomembranes 1778, 1897-1929[3] Refojo, P.N., Sousa, F.L., Teixeira, M., Pereira, M.M. (2010) Biochim Biophys Acta. 2010 Apr 21. [Epub ahead of print]

contactauthor:Inêsa.c.pereIraInstituto de Tecnologia Química e Biológica Universidade Nova de Lisboa, Oeiras, Portugal

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Invitedspeakers 17

oxidative stress, thermogenesis and the evolution of the uncoupling proteinsEduardo Rial, Jesús Jiménez-Jiménez, M. Mar González-Barroso.

Centro de Invetigaciones Biológicas. CSIC, Ramiro de Maerztu 9, Madrid, Spain.

Brown fat (BAT) is a thermogenic tissue used by eutherian mammals to maintain body tem-perature when cold exposed or to burn an excess of calories ingested with the diet. The high thermogenic capacity of the tissue is due to the presence of the uncoupling protein UCP1, a carrier that allows a regulated discharge of the mitochondrial proton gradient leading to fast substrate oxidation without ATP synthesis. The physiological regulation of UCP1 is well es-tablished. Noradrenaline stimulation of the brown adipocyte initiates a lipolytic cascade and the fatty acids released serve two functions: they are substrate for respiration and activators of UCP1 [1].

A growing number of proteins homologous to UCP1 have been described not only in other animal tissues but also in plants or fungi. The ubiquitous presence of the UCPs suggests that physiological uncoupling of oxidative phosphorylation is a general strategy adopted by living organisms to modulate the energetic efficiency. The function of the remaining members of the UCP protein family is not established, but available data point to a role in the antioxidant defence system. The acceleration of respiration due to UCP-mediated uncoupling would lead to a reduction in the production of superoxide. There are many examples where UCPs are upregulated in physiological situations where there is oxidative stress and data suggesting that their presence lowers ROS levels.

Phylogenetic studies and analyses of conservation of syntenic regions demonstrate that there are UCP1 orthologs in mammals, amphibians and fish. Hence, UCP1 is found in vertebrates with and without non-shivering thermogenesis. However, BAT is only present in eutherian mammals and, interestingly, the branch leading to eutherian UCP1 is remarkably long, evi-dencing a strong structural divergence. Indeed, UCP1 from eutherian mammals present two distinct biochemical properties: (1) a high nucleotide-sensitive basal proton conductance in the absence of fatty acids and (2) a high affinity for fatty acids. Therefore the structural divergence was accompanied by a shift in the biochemical properties that fits with the re-quirements for the regulation of thermogenesis [2]. It can be envisaged that eutherian UCP1 evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte [3].

[1] Rial, E., González-Barroso, M.M. (2001) Biochim. Biophys. Acta 1504, 70-81.[2] Jiménez-Jiménez, J., Zardoya, R., Ledesma, A., García de Lacoba, M., Zaragoza, P., González-Barroso, M.M., Rial, E. (2006) J. Mol. Biol. 359,1010-1022.[3] Rial, E., Zardoya, R. (2009) J. Biol. 8, 58.

contactauthor:eduardorIalCentro de Invetigaciones Biológicas. CSIC Ramiro de Maerztu 9, Madrid, Spain.

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Invitedspeakers 18

the physical forces behind collective cell migrationXavier Trepat1,2

1Institut de Bioenginyeria de Catalunya (IBEC)2Facultat de Medicina, Universitat de Barcelona (UB)

Fundamental biological processes including morphogenesis, tissue repair, and tumor metas-tasis require collective cell motions, and to drive these motions cells exert traction forces on their surroundings. The mechanisms underlying this basic principle of health and disease have been debated intensively and, using a variety of methods in vivo, in vitro, and in silico, much conflicting evidence has accumulated. This conflicting evidence has been in every case indirect or inferential, however, because within the moving cell group the physical forces themselves have remained inaccessible to direct experimental observation. Our group has recently obtained by direct measurement the first explicit maps of those physical forces and their distribution. Analysis of these maps revealed that migration of an epithelial cell sheet is not driven by leader cells at the edge of sheet. Instead each cell within the sheet engages in a tug-of-war that integrates local force generation into a global state of tensile stress.

contactauthor:XaviertrepatInstitut de Bioenginyeria de Catalunya (IBEC)

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Invitedspeakers 19

assembling a biological nanomotor on a nano-engineered surfaceC. Vieu, J. Chalmeau, C. Thibault

CNRS ; LAAS ; 7 avenue du colonel Roche, F-31077 Toulouse, FranceUniversité de Toulouse ; INSA, UPS, INP, ISAE ; LAAS ; F-31077 Toulouse, France

Nature offers today thousands of machine at the nano-scale, working in symphony within any living organism on Earth. This basic analysis shows that biology can be seen as a nanoscale phenomenom . Molecular biology, genetics and biochemical methods have been extensively used for studying the struc-ture and principle of these bio-machines from their in vivo observations to their in vitro extraction and purification. These methods have revealed extraordinary capacities, such as for example the F1F0-ATPase . This nano-motor, presents mechanical and biochemical properties, which are for the moment, out of reach to any nano-scale artificial machine produced by silicon technologies. Since the pioneering work of C. Montemagno a new paradigm has thus been proposed; we call it “Nanotechnologies from biology”. The idea is to integrate natural nanoscale bio-machines on devices in order to exploit their exceptional efficiencies. This methodology can be seen as a rupture with respect to the conventional “top-down” and “bottom-up” approaches, because here, the engineering of the active part of the device is devoted to na-ture rather than to human conception.

In this paper we describe the initial technological steps dedicated to the assembly of the flagellar nanomo-tor of bacteria on an artificial engineered surface using nanotechnologies. We will address both questions of the elucidation of the structure and mechanism of this mesoscopic nanomotor and its integration in 2 or 3 dimensions on a solid support. This work is a fundamental research which would serve as a base for the future development of the new area of integrated hybrid bio-inspired devices.

Throug the combination of soft-lithography and self-assembly and using liquid Atomic Force Microscopy for imaging the assembly, we have been able to observe the assembly of one piece of this bionanomachine on a supported phospholipidic bilayer membrane. The structure, the investigation of the interactions bet-ween the constitutive proteins of this machine using Quartz Crystal Microbalance technology (QCM) will be reported together with the possible routes untill the complete assembly of this biological nanomachine involving a combination of Nanotechnologies and synthetic biology.

a) Schematic of the flagellar nanomotor of bacte-ria. The largest ring at the basis (C-Ring) measu-res 45 nm in diameter. b) AFM image in liquid medium of patterned phospholipidic membranes of E. Coli obtained by Micro-Contact printing and selective liposome fu-sion. Scale bar 5 µm.

Figure 2 : AFM image in liquid medium of FliG proteins (MS ring) assembled on a supported phospholipidic mem-branes of E. Coli.

contactauthor:c.VIeuCNRS ; LAAS ; 7 avenue du colonel Roche, F-31077 Toulouse, France

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Invitedspeakers 20

addressing functionally relevant large-scale motions in proteins by molecular-dynamics simulation: the artJ case.Roman Affentranger2 and Xavier Daura1,2

1Catalan Institution for Research and Advanced Studies (ICREA), E-08010 Barcelona.2Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, E-08193 Bellaterra.

Extended access to High Performance Computing (be it large commodity clusters or super-computers) has been, together with algorithm (and code) optimisation and parallelisation, a major component to the recent advancement of biomolecular simulation, even more so than the new developments at the methodological and force field levels have. Despite the specta-cular increase in time scales and system sizes, the capacity to answer questions of biological relevance remains, however, very limited. Model assumptions aside, reaching the system com-plexity and time scale relevant to the process of interest were thought to be the main, if not only, bottleneck. Now that this has been reached for some remarkable biomolecular systems (although still very simple when put on biological perspective), it has become evident that, due to the statistical nature of these processes, a very large number of independent events need to be sampled before arriving to consistent results. Here, we present an example study of protein dynamics that illustrates the importance of statistics: an analysis of the conforma-tional dynamics of a bacterial protein of the Periplasmic-Binding family (PBPs) and its relation to ligand binding.

Acknowledgement:

This project is supported by funding under the Sixth Research Framework Programme of the European Union (ref. LSHB-CT-2006-037325) and by the Spanish Ministry for Education and Science/FEDER (ref. BIO2007-62954). We thank the Barcelona Supercomputing Center (BSC) for computing resources made available through grant BCV-2009-1-0024, and the Port d’Informació Científica (PIC) for providing cluster-computing and data-storage resources.

contactauthor:XavierDauraCatalan Institution for Research and Advanced Studies (ICREA) E-08010 Barcelona.

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Invitedspeakers 21

Biophysical characterization of antimicrobial peptides interaction with model membranes and biological implications on the mechanism of actionMargarida Bastos

CIQ(UP), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Portugal.

Although a significant number of studies have pointed out the exceptional potential of antimi-crobial peptides (AMPs) as encouraging therapeutical agents, their application has proceeded at a slow rate due to the intrinsic limitations of their peptide nature. Therefore, a number of structural modifications leading to enhanced AMP biological lifetimes and therapeutic index have been proposed with different results.

In our Lab, different families of AMPs have been studied by a variety of biophysical techni-ques, such as Differential Scanning Calorimetry (DSC), Isothermal Titration Calorimetry (ITC), Fluorescence Spectroscopy (TRFS), Circular Dichroism (CD), and more recently Small Angle X-Ray Diffraction (SAXD). As model membranes we have been using PC or PC/SM (as models for host erythrocytes and PC/PG or PE/PG as model for bacteria.

The results obtained for 2 families – the Lactoferrin and the Cecropin A-Melittin families- with the above mentioned model membrane systems will be presented and discussed.

The implications of peptide structure and lipid composition on the biophysical profile of the peptide-lipid interactions and their biological activity will be presented and discussed.

Aknowledgements. Thanks are due to FCT for financial support to CIQ(UP), to Prof Jan Bols-cher and Prof David Andreu for providing the peptides and to Prof. Manuel Prieto for access to TRFS instrument.

contactauthor:MargaridaBastosCIQ(UP), Department of Chemistry and Biochemistry Faculty of Sciences University of Porto, Portugal [email protected]

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Invitedspeakers 22

Cellulosome nanomechanics: moving from single cohesin modules to the whole complexMariano Carrión-Vázquez

Instituto Cajal, CSIC, CIBERNED & IMDEA Nanociencia, Avda. Doctor Arce 37, 28002 Madrid

Some nanomachines are based on protein scaffolds to spatio-temporally coordinate a variety of enzymatic activities. The cellulosome is a family of large protein complexes capable of degrading cellulose in a highly efficient manner1. Scaffoldin, the scaffold of this complex, is a modular protein composed a variety of cohesin modules that attaches to the cell and the substrate by a variety of different strategies. The whole complex, as a cell adhesion system, is expected to be subjected to mechanical stress2. In a previous study, we hypothesized that cohesin I modules located between the two anchoring points of scaffoldin (“connecting mo-dules”) might be exposed to mechanical stress and hence would have higher mechanical sta-bility than those outside this region (“hanging” modules). There we showed, by atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations, that connecting modules are the most mechanostable proteins reported to date, while han-ging cohesins present lower stability3. Here we have analyzed, using similar techniques, the mechanical stability of additional connecting and hanging modules The results obtained give further support to our hypothesis. Furthermore, we have studied the effect on the mechanical stability of two elements naturally found in the cellulosome: the natural surrounding linkers located between cohesin I modules and the bound cellulases, both of which may be thought to affect the mechanical stability of cohesins4,5,6. We found no effect of cellulase binding and a slight effect of some linkers. Taken together, our results reinforce our current mechanical model of the cellulosome pointing to the mechanical clamp (i.e., a patch of shear backbone hydrogen bonds) of cohesins as the major molecular determinant of the mechanical barrier. This motif seems to protect the integrity of this module and of its binding surface to cellulases as well as that of the whole complex.

1. Bayer EA., Belaich JP., Shoham Y, Lamed R. (2004) The cellulosomes: Multyenzyme machines for de-gradation of plant cell wall polysaccharides. Annu. Rev. Microbol. 58:521-54.2. Bustamante C., Chemla YR., Forde NR., Izhaky D. (2004) Mechanical processes in biochemistry. Annu. Rev. Biochem. 73:705-48.3. Valbuena A., Oroz J., Hervás R., Vera AM., Rodríguez D., Menéndez M., Sulkowska JI., Cieplak M., Ca-rrión-Vázquez M. (2009) On the remarkable mechanostability of scaffoldins and the mechanical clamp motif. Proc. Natl. Acad. Sci. 18:13791-96.4. Cao Y., Yoo T., Zhuang S., Li H. (2008) Protein-protein interaction regulates proteins’ mechanical sta-bility. J. Mol. Biol. 378:1132-41.5. Politou AS., Gautel M., Joseph C., Pastore A. (1994) Immunoglobulin-type domains of titin are stabili-zed by amino-terminal extension. FEBS letters. 352:27-31.6. Rounsevell RW, Steward A, Clarke J. (2005) Biophysical investigations of engineered polyproteins: implications for force data. Biophys. J. 88:2022-9.

contactauthor:MarianocarrIón-VázquezInstituto Cajal, CSIC, CIBERNED & IMDEA Nanociencia, Avda. Doctor Arce 37, 28002 Madrid email: [email protected]

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Invitedspeakers 23

an optical view of cell membrane compartmentalisation and functional implicationsMaria F. Garcia-Parajo1,2

1 BioNanophotonics group, IBEC- Institute for Bioengineering of Catalonia and CIBER-bbn, Baldiri Reixac 15-21, 08028 Barcelona, Spain2 ICREA- Institució Catalana de Recerca i Estudis Avançats, 0810 Barcelona, Spain

Cells communicate with each other and the outside world through a myriad of receptors expressed at the cell membrane. It has become apparent that these molecules do not operate individually but they are part of complex platforms that organize spatially and temporally in an orchestrated way to accomplish their function. In this presentation I will discuss how op-tical tools designed to work at the nanoscale (NSOM and single particle tracking) can provide valuable information on the spatio-temporal organization of receptors of the immune system in relation to function [1]. Particular topics of interest in our group are the dynamic spatial organization of the integrin receptor LFA-1 mediating cell adhesion and the pathogen recog-nition receptor DC-SIGN and its interaction with the HIV virus.

In the case of the integrin receptor LFA-1 we used a combination of single molecule fluo-rescence techniques to study its spatiotemporal organization on monocytes. We performed optical nanoimaging of LFA-1 nanoclusters in relation to membrane rafts with a resolution of 70nm and accuracy of 3nm [2]. In quiescent cells, LFA-1 does not associate with membrane rafts, independently on the conformational state of the integrin. Moreover, a large population of these nanoclusters diffuses freely on the membrane [3]. Binding of the integrin to its ligand ICAM-1 induces the formation of microclusters that further associate with rafts and exhibit a reduced mobility, consistent with cytoskeleton interactions. Our work highlights the markedly different spatiotemporal organization of LFA-1 that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion.

In the case of the pathogen recognition receptor DC-SIGN we have discovered a large degree of heterogeneous nanoclustering of the receptor on the membrane of immature dendritic cells [4]. This heterogeneity is related to the wide capability of DC-SIGN to recognize a large number of different pathogens, from viruses to bacteria. We are currently following the pa-thways of HIV-virus capture and internalization by applying single molecule techniques in living cells.

[1] T.S. van Zanten et al, (2010) Biochim. Biophys. Acta 1798, 777-787[2] T.S. van Zanten et al, (2009) Proc. Natl. Acad. Sci, USA 106, 18557-18562[3] R. Diez-Ahedo et al., (2009) Small 5, 1258-1263.[4] B. I. de Bakker et al, (2007) ChemPhysChem 8, 1473-1480.

contactauthor:MariaF.garcIa-paraJoBioNanophotonics group, IBEC- Institute for Bioengineering of Catalonia and CIBER-bbn Baldiri Reixac 15-21, 08028 Barcelona, Spain.

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Invitedspeakers 24

Quantification of protein-protein interactions within membranes by fluorescence correlation spectroscopyAna J. García-Sáez1, Jonas Ries2, Mar Orzáez3, Enrique Pérez-Payá3, Petra Schwille2.1Max-Planck for Metals Research and German Cancer Research Center, BioQuant, Im Neuen-heimer Feld 267, 69120 Heidelberg, Germany.2 Depto. Química Médica, Centro de Investigación Príncipe Felipe, Valencia, Spain.3BIOTEC der TU Dresden, Tatztberg 47-51, 01307 Dresden, Germany

The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation [1] or the binding between a growth factor and its receptor during signaling [2].

[1] García-Sáez AJ, Ries J, Orzáez M, Pérez-Payà E, Schwille P. (2009) Nat. Struct. Mol. Biol. 16, 1178-85.[2] Ries J, Yu SR, Burkhardt M, Brand M, Schwille P. (2009) Nat. Methods. 6, 643-5.

contactauthor:anaJ.garcía-sáezMax-Planck for Metals Research and German Cancer Research Center, BioQuant, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany.

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Invitedspeakers 25

Studying protein-ligand interactions in a flexible enzymeP. Martel, V. Ribeiro, V. Teixeira

Center for Molecular and Structural Biomedicine IBB-Institute for Biotechnology and Bioengineering Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8005-114 Faro, Portugal

Cytochromes P450 (CYPs) are extremely versatile enzymes capable of catalyzing a vast num-ber of compounds[1], and play an important role in the elimination of xenobiotics, inclu-ding pharmaceutical drugs[2]. Human CYP3A4 is a particularly important CYP, metabolizing approximately half of the currently marketed drugs, besides endogenous compounds[3] . To metabolize such a variety of compounds, CYP3A4 has to be extremely flexible, which makes interaction studies difficult. We employ a multi-conformational docking setup where confor-mations are sampled from multiple molecular dynamics simulations to analyze the correct binding of various ligands. While productive ligand docking poses are never attained with the receptor x-ray structure, the multi-conformational docking procedure is successful in binding of each ligand in at least one productive conformation. By looking at the docked solutions it is possible to recognize protein residues that may play an important role in ligand access, binding and stabilization[4].

References[1] C.M.Brown, B.Reisfeld, A.N.Mayeno (2008), CytochromesP450: a structure-based summary of bio-transformations using representative substrates, Drug. Metab. Rev., 40:1-100[2] P.Anzenbacher, E.Anzenbacherová (2001), CytochromesP450 and metabolism of xenobiotics, Cell Mol. Life Sci., 58:737-747 [3] F.P.Guengerich (1999), Cytochrome P-450 3A4:regulation and role in drug metabolism, Ann. Rev. Pharm. Toxicol. 39:1-17 [4] V. Texeira, V. Ribeiro, P.J.Martel (2010), Analysis of binding modes of ligands to multiple conforma-tions of CYP3A4, Bioch. Biophys. Acta, in press

contactauthor:p.MartelCenter for Molecular and Structural Biomedicine IBB-Institute for Biotechnology and Bioengineering Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8005-114 Faro, Portugal e-mail: [email protected]

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Invitedspeakers 26

Continuous membrane mechanics: where physics meets cell biologyFrancisco Monroy

Departamento de Química Física I, Universidad Complutense de Madrid, 28040 Madrid, Spain

The physical background on the continuous mechanics of flexible membranes and its im-pact over the classical wisdom of the lipid bilayer is revisited. Then, recent advances on the experimental techniques making available not only membrane mechanics but also dynamics will be presented. These methods have revealed important discrepancies from the classical Helfrich description of the ideal membrane. New experiments and theoretical advances in membrane mechanics will be presented and a modern revision of the lipid bilayer where the internal structure emerges as a central protagonist will be exposed.1-4 As far such a criticism provides new insight on membrane biophysics a new frontier for the quantitative description of biological membranes will be envisaged.

References1. Arriaga et al. “Stiffening effect of cholesterol on disordered lipid phases: a combined NSE + DLS analy-sis of the bending elasticity of large unillamelar vesicles based on POPC, Biophys. J 96, 3629-37 (2009)2. Rodriguez-Garcia et al. Bimodal spectrum for the curvature fluctuations of bilayer vesicles: pure ben-ding plus hybrid curvature-dilation modes, Phys Rev Lett 102, 128101-4 (2009)3. Arriaga et al. “Fluctuation dynamics of spherical vesicles: frustration of regular bulk dissipation into sub-diffusive relaxation” Phys Rev E 80, 031908-1-14 (2009) 4. Arriaga et al. “Domain-growth kinetic origin of nonhorizontal phase coexistence plateaux in langmuir monolayers: compression rigidity of a raft-like lipid distribution” J. Chem. Phys. B, 114, 4509-4520 (2010)

contactauthor:FranciscoMonroyDepartamento de Química Física I Universidad Complutense de Madrid 28040 Madrid, Spain

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Invitedspeakers 27

experimental Studies of primordial protein FoldingVictor Muñoz

Proteins fold into specific three-dimensional structures that determine their biological function in a process that is both spontaneous and reversible. This ability is what sets proteins apart from other heteropolymers, which populate large degenerate ensembles of conforma-tions. The big question is how do proteins accomplish folding during early evolution? And as a subsidiary question: what are the relative contributions from the chemistry of proteins (amino-acid composition) and natural selection (sequence)? In our group we are addressing this question experimentally by studying the conformational properties of synthetic proteins, either designed de novo or obtained by randomizing the sequence of natural proteins. In this talk I will discuss our recent experimental results in this area.

contactauthor:Victor MUñOZ

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Invitedspeakers 28

prion Folding and the Species BarrierTeresa J.T. Pinheiro

Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK

Prion diseases are a group of fatal neurodegenerative disorders that manifest as infectious, sporadic or familial and are all associated with the misfolding of the prion protein (PrP). Disease modulating polymorphisms in the PrP amino acid sequence can make an individual more or less susceptible to infection. Another unique property of prion diseases is the exis-tence of a species barrier, where subtle sequence and folding differences modulates the trans-mission between species. In this talk I will give an overview of the folding mechanism of PrP, highlighting the differences in folding intermediates that can explain the species barrier.

contactauthor:teresaJ.t.pInheIroDepartment of Biological Sciences University of Warwick Gibbet Hill Road, Coventry, CV4 7AL, UK

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Invitedspeakers 29

evolution and engineering of proteinsJose M. Sanchez-Ruiz

Facultad de Ciencias, Departamento de Química Física. Universidad de Granada. 18071-Gra-nada, Spain

Technological and biomedical applications of proteins require modifications (usually muta-tions) that improve one or, more often, several of their properties. Hence, protein engineering is one important branch of biotechnology.

Recent years have witnessed an exponential growth in the number of known protein se-quences. The possibility then arises of effectively guiding protein engineering on the basis of sequence-database analyses that capture how protein properties are shaped during evolution. Two such approaches will be discussed in this talk: A) Screening of combinatorial libraries of protein variants constructed on the basis of sequence-alignment statistics. B) Reconstruction of ancestral proteins.

contactauthor:JoseM.sanchez-ruIzFacultad de Ciencias, Departamento de Química Física Universidad de Granada. 18071-Granada, Spain

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Invitedspeakers 30

Looking at dynamic biofunctional surfaces at the nanoscale: initial stages of bacterial cell divisionMarisel Velez

We have explored at the nanometer scale the role played by the membrane protein ZipA in an-choring FtsZ polymers to the membrane surface and in modulating their dynamic two dimen-sional reorganization. The bacterial protein FtsZ is a soluble GTPase structurally analogous to eukaryotic tubulin and in vivo it assembles on the inner side of the cytoplasmic membrane in the mid region of the bacteria at the time of cell division. The protein self-organizes on the surface after binding to a protein membrane anchor, protein ZipA in the case of E. coli, and tri-ggers the cell division process. It contributes to the recruitment of other proteins participa-ting in forming the protein complex called the divisome. The polymer formed also participates in the force generating process that divides the cell. The dynamic behavior of the polymer observed in vivo is very relevant to its function but it is not understood yet. Planar lipid bila-yers on solid supports are good systems to use as mimics of membrane process because they allow surface characterization at the nanoscale while the proteins remain immersed in buffer solution. We have used simple reconstituted systems accessible to high resolution surface characterization with AFM to explore the effect of lipid composition and the role of different ZipA domains in modulating FtsZ polymerization on surfaces.

contactauthor:Marisel Velez


Short Oral Presentations

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shortoralpresentations

1/Development of a magneto-optical intracellular nanomanipulator: Bringing single-molecule techniques inside the cell. ..................................... 57Adriana M. de Aguilera, Manuel Vázquez, José M. Valpuesta, José L. Carrascosa and J. Ricardo Arias-González,

2/Distinct mechanisms of lipid bilayer perturbation induced by peptides derived from the membrane-proximal external region of hiV-1 gp41 ......... 58Beatriz Apellániz, Shlomo Nir, and José L. Nieva

3/Single-molecule mechanics of rna molecules ............................................ 59Cristiano Bizarro,, Josep Huguet, Felix Ritort,

4/Metal ions and aβ fibrils modulate S100 amyloidogenesis ...................... 60Hugo M. Botelho, Kiran Yanamandra, Günter Fritz, Ludmilla A. Morozova-Roche, Cláudio M. Gomes

5/reexcitations in Cardiac tissue with heterogeneity in repolarization induced by a pulse due to Calcium Current ..................................................... 61Inma R. Cantalapiedra, Angelina Peñaranda, Blas Echebarria, Jean Bragard

6/Cooperativity of single-headed kinesin motors in axonal transport ......... 62Jaume Casademunt, Javier G. Orlandi.

7/nMr Study of arginine rich putative prebiotic peptides ........................... 63Fernando Diez-García, Jorge P. López-Alonso, Miguel A. Pardo-Cea, Douglas V. Laurents, Irene Gómez-Pinto and Carlos González

8/Fluorescence Microscopy Study of Zwitterionic giant unilamellar Liposomes Confined in Sol-gel glasses ........................................................... 64Rocío Esquembre, Sandra N. Pinto, José A. Poveda, Manuel Prieto, C. Reyes Mateo

9/Functional consequences of the voltage-dependent K+ channels immunomodulation in leukocytes ................................................................... 65Joanna Bielanska, Núria Comes, Núria Villalonga, Miren David , Carmen Valenzuela and Antonio Felipe

10/human apoptosis inducing factor (haiF): kinetic characterization of the mitochondrial reaction with naDh .................................................................. 66Patricia Ferreira, Raquel Villanueva, Mª Dolores Miramar, Santos Susin, Mª Luisa Peleato and Milagros Medina M

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11/Simple models in polymer translocations .................................................. 67Alessandro Fiasconaro, Juan José Mazo,, Fernando Falo,

12/the interplay between central carbon metabolism and cofactor balance in tumor cells using 13C metabolic flux analysis: the function of nicotinamide nucleotide transhydrogenase (nnt) ............................................................... 68Paulo Gameiro,, Christian Metallo, Joanne K. Kelleher, and Gregory Stephanopoulos

13/Systems Biology for plant shoot vascular patterning ............................... 70Marta Ibañes, Norma Fàbregas, Ana Confraria, Pau Formosa, Aniuska Bolivar, Joanne Chory, Ana I. Caño-Delgado

14/Structural analysis of Myxococcus xanthus CdnL, a vital protein and member of the CarD-trCF family of bacterial proteins that interact with rna polymerase ........................................................................................................ 71Yasmina Mirassou, Diana García-Moreno, Clara M. Santiveri, Jorge Santoro, Montserrat Elías-Arnanz, S. Padmanabhan, M. Angeles Jiménez

15/Direct Visualization of the oligomerization properties of proteins by in situ atomic Force Microscopy: the Case of the Ferric uptake regulator Fura analyzed under Different redox Conditions ................................................... 72Anabel Lostao,, María Luisa Peleato, Andrés González, Carlos Gómez-Moreno, and María F. Fillat

16/Facilitative glucose transporter gLut12: novel data showing electrogenic properties ........................................................................................................... 73Alejandra Pérez, Jonai Pujol, Alejandro Reyes, M. Pilar Lostao

17/positioning water molecules around the Mn-Cluster of photosystem ii from pulsed epr experiments. ......................................................................... 74Jesús I. Martínez, Pablo J. Alonso, Inmaculada Yruela, Rafael Picorel

18/Conjugated Linoleic acid impairs adipose plasma Membrane permeability and Fluidity ................................................................................. 75Ana P. Martins, Paula A. Lopes, Susana V. Martins, Ana Madeira,, Nuno C. Santos, José A. M. Prates, Teresa F. Moura, Graça Soveral,

19/Functional integration of the portal protein of bacteriophage Φ29 in lipid membranes ...................................................................................................... 76Lara H Moleiro, Ileana Márquez, Sonia Moreno, Jose L Carrascosa, Iván López-Montero, Marisela Vélez and Francisco Monroy

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20/tools-4-Metatool. online suite of web-tools to process stoichiometric network analysis data from MetatooL .......................................................... 77Federico Morán, Francisco Montero, Sara Vázquez, Daniela Xavier, Alessio Gamba, Paola Bettinelli.

21/a molecular simulation study of the activity of anesthetics molecule on biological membrane of different lipidic composition .................................. 78Sara D. Oliveira Costa, Rodolfo D. Poraso and J. J. López Cascales

22/Structure of the plakin domain of plectin. ................................................. 79Esther Ortega Portero , Rubén Martínez Buey, , Arnoud Sonnenberg, Jose Mª de Pereda

23/insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC ............................................................................... 80Inmaculada Pérez-Dorado,, Ana González,, María Morales,, Reyes Sanles, Waldemar Striker,, Waldemar Vollmer,, Shahriar Mobashery, Jose Luis García, Martín Martínez-Ripoll, Pedro García,, Juan Antonio Hermoso

24/rotational dynamics of yeast ribosomal stalk proteins in living cells by multiphoton time-resolved Fluorescence polarization micro-spectroscopy 81Sebastian Raja, Carolina García, Miguel A.Sacristán, Juan P. G. Ballesta, M.Pilar Lillo

25/Functional characteristics of synaptic vesicles at active Zones ............... 82Rocío Ruiz, Raquel Cano, William J. Betz and Lucía Tabares

26/principles of Quantitative evolutionary Design of redox Cycles ............. 83Armindo Salvador,, Pedro Coelho,,, Bharathi Pandurangan, Michael A. Savageau

27/the role of synaptotagmin 7 in the release of catecholamines from adrenal chromaffin cells .................................................................................... 84Margarita Segovia, Anton Maximov,, Thomas C. Südhof, & Guillermo Álvarez de Toledo

28/Characterization of the regions involved in the calcium-induced folding of the intrinsically disordered rtX motifs from Bordetella pertussis adenylate cyclase toxin....................................................................................................... 85Ana-Cristina Sotomayor Pérez, Johanna Karst, Marilyne Davi, Iñaki Guijarro, Daniel Ladant, Alexandre Chenal.

29/anionic Currents in pollen protoplasts from Lilium longiflorum ............. 86Bárbara Tavares,,, Pedro Nuno Dias,,, Teresa Fonseca Moura, José Alberto Feijó, and Ana Bicho

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30/prediction of solution properties of fully and partially, intrinsically disordered proteins ........................................................................................... 87Diego Amorós, Álvaro Ortega and José García de la Torre

57


shortoralpresentations 1

Development of a magneto-optical intracellular nanomanipulator: Bringing single-molecule techniques inside the cell.Adriana M. de Aguilera1,2, Manuel Vázquez3, José M. Valpuesta2, José L. Carrascosa2 and J. Ricardo Arias-González1,2

1IMDEA Nanociencia, 28049 Madrid, Spain2Centro Nacional de Biotecnología, CSIC, 28049 Madrid, Spain3Instituto de Ciencia de Materiales de Madrid, CSIC, 28049 Madrid, Spain

Single-molecule techniques, such as AFM and optical and magnetic tweezers, have shed light on the physical properties and function of many relevant biological molecules, such as DNA elasticity or the force involved on each kinesin step [1]. We aim to transfer this novel appro-ach in the study of biological problems inside the cell. Specifically, studying the cell’s reaction to a physical change that is exerted on any of its components will shed understanding on how physics interplay their cellular role. Optical tweezers lack specificity for manipulation purposes inside the cell [2] since all the cell components are dielectric. Magnetic fields in contrast provide high specificity because none of the biological structures of most cells are magnetic [3]. Our approach involves the use of magnetic nanoparticles attached to single molecules or single organelles, such as the centrosome [4]. Magnetic forces can be generated by assembling electrochemically-sharpened amorphous ferromagnetic microwires [5] inside custom-designed microfluidics chambers. The microwires in turn will be fed by computer-controlled electromagnets. A theoretical treatment of the magnetic field and computational simulations by the finite element method of the field within the microwire-tips are used to predict the forces involved in the experiment. Magnetic forces thus generated will allow the manipulation of magnetic nanoparticles inside a living cell. An optical trap will be used to select, move and hold the individual cell within the microwire-tips. This approach seeks to un-derstand the physical basis of key biological processes, such as cell division and organization, and its immediate link to uncontrolled proliferation, which is important in tumor genesis [6].

References[1] C. Bustamante, J. C. Macosko, G.J. Wuite (2000). Nat Rev Mol Cell Biol 1(2): 130-6.[2] S. Hormeno, and J. R. Arias-Gonzalez (2006). Biol Cell 98(12): 679-95.[3] A.H.B. de Vries, B.E. Krenn, R. van Driel and J.S. Kanger. (2007) Biophysical Journal, 88 2137-2144.[4] S. Hormeño, B. Ibarra, F.J. Chichón, K. Habermann, B.M.H. Lange, J.M. Valpuesta, J.L. Carrascosa, J.R. Arias-Gonzalez. (2009) Biophysical Journal, 97 1022-1030.[5] M. Vázquez, “Advanced Magnetic Microwires” in Handbook of Magnetism and Advanced Magnetic Materials. H. Kronmüller & S. Parkin, Ed. 4 (2007), Novel Materials. John Wiley & Sons, Ltd. ISBN: 978-0-470-02217-7[6] E. Rebollo, P. Sampaio, J. Januschke, S. Llamazares, H. Varmark, C. Gonzalez. (2007). Develop. Cell 12, 467-474.

contactauthor:adrianaM.deaguIleraIMDEA Nanociencia, 28049 Madrid, SpainCentro Nacional de Biotecnología, CSIC, 28049 Madrid, Spain

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Distinct mechanisms of lipid bilayer perturbation induced by peptides derived from the membrane-proximal external region of hiV-1 gp41Beatriz Apellániz1, Shlomo Nir2, and José L. Nieva1

1Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain. 2Seagram Center for Soil and Water Sciences, Faculty of Agricultural, Food and Environmen-tal Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

The conserved, membrane-proximal external region (MPER) of the human immunodeficiency virus type-1 envelope glycoprotein 41 subunit is required for fusogenic activity. It has been proposed that MPER functions by disrupting the virion membrane. Supporting its critical role in viral entry as a membrane-bound entity, MPER constitutes the target for broadly neutra-lizing antibodies that have evolved mechanisms to recognize membrane-inserted epitopes. We have analyzed here the molecular mechanisms of membrane permeabilization induced by N-preTM and PreTM-C, two peptides derived from MPER sequences showing tendency to associate with the bilayer interface or to transfer into hydrocarbon-core, respectively. Both peptides contained the full epitope sequence recognized by the 4E10 monoclonal antibo-dy (MAb4E10), which was subsequently used to probe peptide accessibility from the water phase. N-preTM and PreTM-C capacities for associating with vesicles and inducing their per-meabilization were comparable. However, MAb4E10 specifically blocked the permeabilization induced by N-preTM, but did not appreciably affect that induced by PreTM-C. Supporting the existence of different membrane-bound structures, N-preTM run as a monomer on SDS-PAGE and induced graded release of vesicular contents, while PreTM-C migrated on SDS-PAGE as dimers and permeabilized vesicles following an all-or-none mechanism. These results support the functional segmentation of gp41 membrane regions into hydrophobic subdomains, which might induce distinct membrane-disrupting effects during the fusion cascade.

contactauthor:BeatrizapellánIzUnidad de Biofísica (CSIC-UPV/EHU) and Departamento de BioquímicaUniversidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain

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Single-molecule mechanics of rna moleculesCristiano Bizarro1,2, Josep Huguet1, Felix Ritort1,2

1Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, Avinguda Diagonal 647, 08028 Barcelona, Spain2CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, Spain

The ability to mechanically manipulate individual biomolecules in single molecule experi-ments give us the possibility to extract information about the thermodynamics and kinetics of biological processes without the ensemble averaging of bulk experiments. By using optical tweezers to mechanically unzip single DNA molecules, together with statistical physics me-thods developed in our laboratory, we were able to measure the nearest_neighbor base-pair free energies with great precision at different experimental conditions. In order to extend our studies to RNA molecules, we devised a strategy to synthesize in vitro large RNA hairpins containing 2kbps in length. By pulling apart the two strands of a large RNA hairpin and moni-toring in real time the forces associated with the sequential disruption of base pairs from the double stranded structure, sequence-dependent force-distance curves (FDCs) were obtained. Differently from DNA pulling experiments, in which essentially reversible FDCs were obtained at low pulling rates (allowing us to estimate equilibrium FDCs), in RNA pulling experiments we obtained FDCs containing regions of hysteresis.

To tackle this problem, we are currently applying a method developed in our laboratory[1] for determining the free energy of coexisting states from the irreversible work measurements to these particularly hysteretic regions of RNA FDCs. We have performed many pulling cycles restricted to each one of the selected regions in order to give us sufficient data to characterize accurately the intermediate states associated with each region.

[1] SJunier, I., Mossa, A., Manosas, M. and Ritort, F. (2009) Phys. Rev. Lett. 102, 070602.

contactauthor:cristianoBIzarroDepartament de Física Fonamental, Facultat de Física Universitat de Barcelona Avinguda Diagonal 647, 08028 Barcelona, Spain

CIBER de Bioingeniería, Biomateriales y Nanomedicina Instituto de Salud Carlos III, 28029 Madrid, Spain

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Metal ions and aβ fibrils modulate S100 amyloidogenesisHugo M. Botelho1, Kiran Yanamandra2, Günter Fritz3, Ludmilla A. Morozova-Roche2, Cláudio M. Gomes1

1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157 Oeiras, Portugal. 2Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.3University of Freiburg, Department of Neuropathology, Freiburg, Germany.

Protein deposition as amyloid oligomers in the brain is the cytological hallmark of neurodege-nerative diseases including Alzheimer’s and Parkinson’s. The identification of cellular modula-tors of protein deposition remains a challenging issue. In this respect, the recently identified amyloidogenic properties of S100 proteins opens new possibilities in regard to the possible implication of these proteins in neurodegenerative processes [1]. S100 proteins regulate cell cycle progression, cell growth, differentiation and mobility in vertebrates in a tissue-specific manner. They have rich signal transducing properties, binding calcium and, in some cases, zinc, copper and/or magnesium.

Metal ions modulate S100 conformation: this involves Ca2+ binding effects at the EF-hands and interactions with secondary sites, such as Zn2+ sites found for example in S100A2. In the latter, metal ions affect protein stability, Zn2+ destabilizing and Ca2+ stabilizing it [4]. The effect of metal ions in S100 conformations was also recently pointed out in new amyloidoge-nic processes in the S100A8/A9 proteins [1]. These form metal-mediated amyloid structures, identical to those found in proteinaceous inclusions associated with pre-carcinogenic infla-mmatory foci in the prostate named corpora amylacea, under physiological-like conditions.

Here we report groundbreaking data regarding amyloid formation by S100 proteins overex-pressed in the brain in amyloid diseases (Alzheimer’s and ALS): S100A6, S100A12 and S100B [2,3,5,6]. These proteins form amyloid-like structures characterized by the typical Thioflavin T fluorescence and FT-IR fingerprints. We have screened the effect of calcium and zinc, two major players in the chemical biology of the glutamatergic synapse, in the amyloidogenesis pathway, finding a strong dependence on the formation kinetics and final structure, according to AFM analysis. More interestingly, preformed Aβ fibrils, the ones found in the brain of Alzheimer’s patients, seed amyloid formation by S100B, one of the most abundant proteins in the brain. In addition, S100 amyloid-like species exhibit enhanced cytotoxicity in the oligome-ric, pre-fibrillar state. Overall, our data suggest a cross-talk between Aβ and S100 deposition, cytotoxicity and neurodegeneration.

[1] Yanamandra, K., et al. (2009) PLoS One 4, e5562 [2] Hoyaux, D., et al. (2002) J Neuropathol Exp Neurol 61, 736-744 [3] Mrak, R. E., et al. (2001) Neurobiol Aging 22, 915-922 [4] Botelho, H. M., et al. (2009) FEBS J 276, 1776-1786 [5] Boom, A., et al. (2004) Biochim Biophys Acta 1742, 161-168 [6] Shepherd, C. E., et al. (2006) Neurobiol Aging 27, 1554-1563

contactauthor:hugoM.BotelhoInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Av. da República, EAN, 2780-157 Oeiras, Portugal

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reexcitations in Cardiac tissue with heterogeneity in repolarization induced by a pulse due to Calcium CurrentInma R. Cantalapiedra1, Angelina Peñaranda1, Blas Echebarria1, Jean Bragard2

1Departament de Física Aplicada, Universitat Politècnica de Catalunya, Av. Dr. Marañón 44-50, E-08028 Barcelona.2Departamento de Física y Matemática Aplicada, Universidad de Navarra, Ed. “Los Castaños”, Facultad de Ciencias, 31080 Pamplona

Ventricular fibrillation (VF) underlies most cases of sudden cardiac death. Typically it is pre-ceded by a state of ventricular tachycardia (VT), generated by high frequency reentrant waves (rotors). Transition from VT to VF involves the breakup of a single rotor into multiple wave-lets, generating a state of turbulent electrical activity in the ventricles that impedes cardiac contraction. Reentry occurs when a propagating impulse fails to die out after normal car-diac activation but persists to reexcite the heart after the expiration of the refractory period. We consider that the arrhythmogenic substrate is a dispersion in epicardial cells repolariza-tion[1-2]. To reproduce reexcitations observed in Brugada syndrome, we analyze the effect of a variation in the dynamics of Na+ inactivation and a heterogeneous rise of transient outward current in epicardiac tissue in simplified ionic model. With the help of this model, we analyze the conditions that result in domeless action potentials (APs), as well as possible mechanisms for reexcitation in a cable. We then study the induction of reentrant waves (spiral waves) in simulations of AP propagation in two-dimensional tissue and in the ventricles. We show that reexcitation takes place via a slow pulse due to calcium current that propagates into the region of short APs until it encounters excitable tissue. We calculate analytically the speed of this pulse, and give an estimate of the minimal tissue size necessary for reexcitation.

[1] Antzelevitch C. (2007). Am J Physiol Heart Circ Physiol, 293 H2024-H2038.[2] Miyoshi S, Mitamura H, Fujikura K, Fukuda Y, Tanimoto K, Hagiwara Y, Ita M, Ogawa S. (2003) Am J Physiol Heart Circ Physiol. 284, H1285- H1294.

contactauthor:Inmar.cantalapIedraDepartament de Física Aplicada Universitat Politècnica de Catalunya Av. Dr. Marañón 44-50, E-08028 Barcelona.

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Cooperativity of single-headed kinesin motors in axonal transportJaume Casademunt, Javier G. Orlandi.

Departament d’Estructura i Constituents de la Matèria, Universitat de Barcelona

Recent studies have discovered a remarkable degree of cooperativity of molecular motors based on a noise-driven ratchet mechanism [1], such as single-headed kinesin (KIF1A). This enhanced cooperativity is manifest when motors perform tasks that require groups of motors and in which the external force is unevenly distributed. Examples of these are the formation of the endoplasmic reticulum tubular network or the intracellular vesicle transport. While two-headed kinesin motors are ubiquitous in most cells, single-headed kinesins have been found specifically in axonal transport of neurons, even though single-headed kinesin is a priori a relatively inefficient motor when taken individually. The reason for this specificity in not known. We pursue the hypothesis that the particular adaptation of single-headed kinesins to collective performance is at the root of their specificity to axonal transport, which requires long ranged processivity, high speeds and large forces. Understanding collective behaviour of KIF1A motors could be directly relevant to several neurodegenerative diseases that have been associated to axonal traffic disorders. To this aim, we study the collective behaviour of motors under the above conditions with a two-state ratchet model with different motor-motor interactions, including the possibility that the cohesion of motor clusters, which favours coo-perativity, is controlled by membrane rafts, as suggested by some experiments. Numerical simulations show that the collective behaviour of motor clusters can indeed explain the en-hanced collective performance of KIF1A motors in general, and some intriguing phenomena such as the reversal of the velocity of long tubular cargoes.

[1] Brugués, J., Casademunt, J. (2009) Phys. Rev. Lett. 102, 118104. [2] Campàs, O., Leduc, C., Bassereau, P., Casademunt, J., Joanny, J.-F., and Prost, J., (2008) Biophys. J. 94, 5009–5017.

contactauthor:JaumecasadeMuntDepartament d’Estructura i Constituents de la Matèria Universitat de Barcelona

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nMr Study of arginine rich putative prebiotic peptidesFernando Diez-García1, Jorge P. López-Alonso1, Miguel A. Pardo-Cea1, Douglas V. Laurents1, Irene Gómez-Pinto1 and Carlos González1

1Departamento de Química Física Biológica, Instituto de Química-Física “Rocasolano”, Conse-jo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid.

KIA peptides are twenty residue peptides, composed only of Ala, Gly, Ile, Lys and a C-terminal aromatic residue, that tetramerize to form well folded four helix bundle proteins [1]. These amino acids form under putative prebiotic Earth conditions and KIA peptides weakly interact

with RNA and, in presence of certain diva-lent cations, can promove the cleavage of single-stranded regions in an RNA hairpin [2]. This has led us to hypothesize that well-folded proteins might have co-inhabited the “RNA-World” [1] and based on the superior RNA binding capacity of Arg-rich motifs (ARMs) [3], we envisage that the substitu-tion of Lys by Arg would improve the capa-city of these peptides to recognize and inte-ract with RNA. We have described the structure and conformational stability of these newly designed peptides, called RIA7. Arginine forms under putative prebiotic conditions and should favor the RNA inte-raction as the guanidium moiety of the side

chain, has a greater capacity to donate H-bonds to phosphate groups and form superior cation-π interactions with the nitrogenated bases [4]. By CD experiments, denaturation as-says, hydrogen exchange measurements and NMR spectroscopy, it has been shown that RIA7 peptides are as stable as Lys variants [1] and tetramerize to adopt four-helix bundle structu-res. These peptides have a well packed hydrophobic core composed of the Ile, Tyr and some Ala residues, while the Arg side chains are located on the hydrophilic faces of the peptides and are fully exposed to solvent (Figure). Future experiments will allow us to test the RNA-binding and RNA-cleavage capacities of RIA7.

[1] López de la Osa, J., Bateman, D. A., Ho, S., González, C., Chakrabartty, A. and Laurents, D. V. (2007) Proc. Natl. Acad. Sci. 104, 38, 14941–14946.[2] López-Alonso, J. P., Pardo-Cea, M. A., Gómez-Pinto, I., Fernández, I., Chakrabartty, A., Pedroso, E., González, C. and Laurents, D. V. (2010) Chem. Eur. J., 16, 5314–5323.[3] Weiss, M. A. and Narayana, N. (1998) Biopoly, 48, 167-180.[4] Doughetry, D. A. (1996) Science 271, 163-168.

contactauthor:FernandodIez-garcíaDepartamento de Química Física Biológica, Instituto de Química-Física “Rocasolano” Consejo Superior de Investigaciones Científicas Serrano 119, 28006 Madrid.

Figure.- Previous structure of RIA7: four helix-bundle with showing buried Ile (blue) and Tyr (green) and exposed Arg (red) residue.

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Fluorescence Microscopy Study of Zwitterionic giant unilamellar Liposomes Confined in Sol-gel glassesRocío Esquembre1, Sandra N. Pinto2, José A. Poveda1, Manuel Prieto2, C. Reyes Mateo1 1 Instituto de Biología Molecular y Celular. Universidad Miguel Hernández de Elche 03202 Elche (Alicante), Spain.2 Centro de Química-Física Molecular. Instituto Superior Técnico, Av. Rovisco Pais, P-1049-001, Lisboa, Portugal.

Immobilization of cells or artificial liposomes on solid supports shows interesting applications in protein biology, membrane biophysics, biomedicine, biosensor technology and new mate-rials developing. One of the most successful immobilization method for biological systems is the encapsulation in silica matrices prepared using the sol-gel process. In a recent work we have characterized the biophysical properties of zwitterionic liposomes entrapped in this type of glasses by steady-state and time-resolved fluorescence spectroscopy. Results showed that upon encapsulation, the thermodynamic properties as well as the fluidity of the lipid bilayer exhibited alterations which could limit the use of these systems for practical applications [1]. Nevertheless, this study did not provide direct information about the effect of encapsulation at the level of a single vesicle. In order to get more insight into this matter, microscopy ex-periments have been carried out by immobilizing for the first time giant unilamellar vesicles (GUVs) in a sol-gel matrix. In this study GUVs prepared by electroformation of single or binary mixtures of zwitterionic phospholipids and immobilized in a sol-gel matrix has been characte-rized by making use of several fluorescence microscopy approaches (confocal, two-photon ex-citation fluorescence microscopy and fluorescence correlation spectroscopy-FCS) combined with different fluorescent probes (Rhd-DOPE, LAURDAN and Bodipy-PC). By these techniques we have explored the morphology, domain coexistence, phase state, packing and lipid mobili-ty of the immobilized systems. Results show that the immobilization process affects the mean size of the GUVs, showing a noticeably decrease of the GUVs radius and often a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation; moreover the immobilization process slightly increases the proportion of gel phase in binary lipid mixtures where both gel and fluid phases coexist. Although Laurdan GP images do not show relevant differences between GUVs in solution and immobilized, FCS experiments has revealed that lipid lateral diffusion decreases upon sol-gel entrapment, probably due to the establishment of interactions between the polar head of the zwitterionic phospholipids and the negative-charged silica surface of the porous matrix.

[1] Esquembre, R., Ferrer, M.L., Gutiérrez, M.C., Mallavia, R. y Mateo, C.R. Journal of Physical Chemistry B, 111 (14): 3665-3673, 2007

contactauthor:rocíoesqueMBreInstituto de Biología Molecular y Celular Universidad Miguel Hernández de Elche 03202 Elche (Alicante), Spain.

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Functional consequences of the voltage-dependent K+ channels immunomodulation in leukocytesJoanna Bielanska1, Núria Comes1, Núria Villalonga1, Miren David 2, Carmen Valenzuela2 and Antonio Felipe1

1Molecular Physiology laboratory, Departament de Bioquímica i Biología Molecular, Institut de Biomedicina, Universitat de Barcelona. Avda Diagonal 645, E-08028 Barcelona, Spain. 2Instituto de Investigaciones Biomédicas “Alberto Sols”, CSIC/UAM. C/Arturo Duperier 4, E-28029 Madrid, Spain

Voltage-dependent potassium channels play a pivotal role in the modulation of leukocyte physiology. Myeloid cells are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of Kv channels by specific antagonists decreases cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of po-tassium channel functions in the immune response to infection and inflammation. Here we demonstrate that the biophysical properties of voltage-dependent K+ currents are modified upon activation or immunosuppression in leukocytes. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric Kv1.3/Kv1.5 channels, which generate the main Kv in macrophages. An increase in K+ current amplitude in LPS-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumu-lative inactivation and a more effective Margatoxin inhibition than control cells. These bio-physical parameters are related to an increase in Kv1.3 subunits in the Kv1.3/Kv1.5 hybrid channel. In contrast, DEX decreased the C-type, the cumulative inactivation and the sensitivity to Margatoxin concomitantly with a decrease in Kv1.3 expression. Neither of these treatments apparently altered the expression of Kv1.5. In addition, shRNA lentiviral infection against Kv1.3 effectively blocked the effects. Our results demonstrate that the immunomodulation of leukocytes triggers molecular and biophysical consequences in Kv1.3/Kv1.5 hybrid channels by altering the subunit stoichiometry.

Supported by BFU2008-00431 and CSD2008-00005 from MICINN (Spain)

contactauthor:Joanna BIELANSKAMolecular Physiology laboratory, Departament de Bioquímica i Biología Molecular Institut de Biomedicina, Universitat de Barcelona Avda Diagonal 645, E-08028 Barcelona, Spain.

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human apoptosis inducing factor (haiF): kinetic characterization of the mitochondrial reaction with naDhPatricia Ferreira1, Raquel Villanueva1, Mª Dolores Miramar1, Santos Susin2, Mª Luisa Peleato1 and Milagros Medina M1

1Departamento de Bioquímica y Biología Molecular y Celular and Instituto de Biocomputa-ción y Física de Sistemas Complejos. Universidad de Zaragoza, Spain.2Centre de Recherche des Cordeliers. Paris, France.

AIF is a flavoenzymes with ubiquitous distribution in eukaryotes that shares homology with different families of oxidoreductases. AIF is released from the mitochondrial intermembrane space and translocates to the nucleus where it causes chromatin condensation and DNA frag-mentation. Therefore, AIF participates in a route of programmed cellular death independent of caspases [1]. AIF also shows a NADH dependent oxidoreductase activity through its FAD cofactor, existing certain controversy on its relationship with the role of AIF in apoptosis [2]. This redox activity has also been related to the integrity of some protein complexes of the electron transport chain, but so far its redox protein partner in the cellular environment is unknown. We are currently characterizing the AIF redox function in mitochondria. In order to identify the characteristics of the in vivo AIF electron acceptor, different chemical compounds and redox proteins are being tested in accepting the electrons from hAIF when is reduced by NADH. The hydride transfer mechanism from NADH to hAIF has been investigated using pre-steady state fast kinetics. The hAIF reduction by NADH occurs through protein dimerization and charge transfer complexes formation, in agreement to the data reported by the mouse enzyme [2, 3]. These complexes are very stables and its production prevents hAIF reoxidation by molecular oxygen, indicating that molecular oxygen is not the natural electron acceptor of AIF. The hAIF crystal structure suggests that several residues in the flavin environment which could be modulating the substrate interaction and the hydride transfer processes (P173, K177, F310 and W483). The role of these residues has been investigated by site directed mutagenesis. The biomedical use of AIF, including the screening of inhibitors or activators, requires of the understanding of its action mechanism. Our studies on hAIF will surely contri-bute to identify the AIF electron acceptor, as well as to clarify if the redox function is linked to apoptosis.

[1] Lipton, S. A. (2002). Cell. 111, 147-150[2] Miramar, M. D. (2001). J Biol Chem. 276, 16391-16398[3] Sevrioukova, I. F. (2009). J Mol Biol. 390, 924-938

contactauthor:patriciaFerreIraDepartamento de Bioquímica y Biología Molecular y Celular and Instituto de Biocomputación y Física de Sistemas ComplejosUniversidad de Zaragoza, Spain.

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Simple models in polymer translocationsAlessandro Fiasconaro1, Juan José Mazo1,2, Fernando Falo1,3

1Departamento de Física de la Materia Condensada, Universidad de Zaragoza, Spain. 2Instituto de Ciencia de Materiales de Aragón CSIC-Universidad de Zaragoza, Spain.3Instituto de Biocomputación y Física de Sistemas Complejos, Universidad de Zaragoza, Spain.

Transport mechanism of molecules inside cells and/or through cell membranes is getting nowadays more and more attraction. On one hand, because of the increasing abilities to de-tect and to measure the biological mechanisms at the nanoscale, on the other hand, because of the challenging possibility to construct from scratch structures (with both natural and synthetic materials) able to imitate the biological functioning [1].

Long molecule translocation is usually driven by constant fields in the pore or by chemical po-tential differences in both sides of the membrane. In other cases the translocation is assisted by an ATP-based molecular motor [2].

Goal of this work is to present the model of a simple motor with different driving mechanisms. The model is able to describe the polymer translocation in one dimension and our results agree qualitatively with recent experimental outcomes on DNA packaging in bacteriophage [3].

[1] Mickler M. Schleiff, E., and Hugel T. (2008) Chem. Phys. Chem. 9, 1503-1509.[2] Hanggi P., Marchesoni F., (2009) Rev. Mod. Phys, 81, 683-693. [3] SMITH D. E., TANS S. J., SMITH S. B., GRIMES S., ANDERSON D. L., AND BUSTAMANTE C., (2001) Nature 413, 748.

contactauthor:alessandroFIasconaroDepartamento de Física de la Materia Condensada Universidad de Zaragoza, Spain

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the interplay between central carbon metabolism and cofactor balance in tumor cells using 13C metabolic flux analysis: the function of nicotinamide nucleotide transhydrogenase (nnt)Paulo Gameiro1,2, Christian Metallo1, Joanne K. Kelleher1, and Gregory Stephanopoulos1

1Chemical Engineering Department, Massachusetts Institute of Technology, 77 Mass Ave, 56-439, Cambridge, MA 02139, USA2Life Sciences Department, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal

During proliferation a cell undergoes fundamental changes in energy metabolism prior to di-vision. In particular, tumor cells increase aerobic glycolysis [1] and the pentose phosphate pa-thway to produce biosynthetic precursors, NADPH and lactate, which recycles cytosolic NAD+ and facilitates invasion in vivo [2],[3]. In contrast, the tricarboxylic (TCA) cycle is functionally coupled to oxidative phosphorylation (OXPHOS). In this context, glutamine – derived anaple-rosis is necessary for tumor cells to proliferate in vitro [4]. Yet, less is known about the signifi-cance of mitochondrial cofactors to synchronize TCA cycle activity in tumor cells. In addition, an emerging theme in cancer biology is how tumor cells maintain an adequate supply of mitochondrial NADPH, which prevents the overproduction of mitochondrial reaction oxygen species (ROS). Thus, we have anticipated that a perturbation of cofactor balance would impair the central carbon metabolism of highly proliferative cells.

In the present study, we have conducted metabolic flux analysis (MFA) using 13C glucose and glutamine tracers to evaluate the role of Nicotinamide Nucleotide Transhydrogenase (NNT) in two cancer cell lines that display distinct metabolic features: a Warburg – like cell line - A549, and a malignant melanoma - Sk-Mel5. NNT is a mitochondrial enzyme that makes use of the mitochondrial membrane potential to reversibly recycle NAD+ within the matrix with the concomitant production of NADPH: H+out + NADH + NADP+ ↔ H+in + NAD+ + NADPH [5].

NNT has been implicated in glucose homeostasis and insulin secretion [6] but its role in can-cer metabolism is unknown. Herein, we demonstrate that shRNA – mediated knockdown of NNT i) affects the steady-state activity of TCA cycle and synthesis of fatty acids, ii) causes ATP deficiency, and iii) reduces the levels of reduced glutathione. The present findings ascribe a main role to NNT in synchronizing the NADH/NAD+ equilibrium with TCA cycle and OXPHOS, and maintaining the mitochondrial reducing power that copes with metabolic stress. Our results suggest that NNT may be a buffering system whose activity and directionality are ne-cessary for proliferation and may be mechanistically dependent on the metabolic phenotype of a tumor cell.

[1] Warburg, O. (1956) Science 123, 309-314.[2] Weinberg, F., Hamanaka, R., Wheaton, W.W., Weinberg, S., Joseph, J., Lopez, M., Kalyanaraman, B., Mutlu, G.M., Budinger, G.R.S., Chandel, N.S. (2010) PNAS 107, 8788-93.[3] Gatenby R.A., and Gillies, R.J.(2008) Nat Rev Cancer. 8, 56-61.[4] Kaadige, M.R., Looper, R.E., Kamalanaadhan S., and Ayer, D.E. (2009) PNAS 106, 14878–14883.[5] Rydström, J. (2006) Biochimica et Biophysica Acta 1757, 721–726.[6] Freeman, H., Shimomura, K., Horner, E., Cox, R.D., Ashcroft, F.M. (2006) Cell Metabolism 3, 35-45.

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contactauthor:paulogaMeIroChemical Engineering DepartmentMassachusetts Institute of Technology77 Mass Ave, 56-439, Cambridge MA 02139, USA

Life Sciences DepartmentFaculty of Sciences and TechnologyUniversity of Coimbra, Coimbra, Portugal

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Systems Biology for plant shoot vascular patterningMarta Ibañes1, Norma Fàbregas2, Ana Confraria2, Pau Formosa1, Aniuska Bolivar1, Joanne Chory3, Ana I. Caño-Delgado2

1Departament Estructura i Constituents de la Matèria, Universitat de Barcelona, Diagonal 647, 08028 Barcelona, Spain.2Molecular Genetics Department. Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB), C/ Jordi Girona Salgado 18-24, 08034 Barcelona, Spain.3Howard Hughes Medical Institute and Plant Biology Laboratory, The Salk Institute for Biolo-gical Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037

The plant vascular system provides transport and support capabilities that are essential for plant growth and development. This system, which connects distant organs within a plant, is organized in a variety of different patterns. How do these patterns emerge? The mechanisms driving the venation patterns that vascular tissues form in leaves have received a lot of at-tention from theoretical and computational points of view. However, little is known on how the vascular pattern in shoots arises. In the inflorescence stem of plant model Arabidopsis thaliana, the vascular tissue is organized in bundles, forming periodic patterns along a ring when observed in shoot cross-sections. Here we show both computationally and experimen-tally how we can alter this periodic pattern [1]. Our results suggest that the polar transport of plant hormone auxin, and not overall auxin levels, drive periodic auxin maxima that position vascular bundles. In addition, we find that plant steroid hormones brassinosteroids dictate the size of the field of cells on which the pattern arises, modulating vascular bundle number. We expect that these results, together with recent knowledge acquired on other patterning pro-cesses like leaf venation and lateral organ positioning, will foster future research on design principles of plant patterning and development [2].

[1] Ibañes M, Fàbregas N, Chory J, Caño-Delgado AI. PNAS 106(32):13630-5 (2009).[2] Fàbregas N, Ibañes M, Caño-Delgado AI. Plant Signaling & Behaviour (in press).

contactauthor:MartaIBañesDepartament Estructura i Constituents de la Matèria Universitat de Barcelona Diagonal 647, 08028 Barcelona, Spain.

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Structural analysis of Myxococcus xanthus CdnL, a vital protein and member of the CarD-trCF family of bacterial proteins that interact with rna polymeraseYasmina Mirassou1, Diana García-Moreno2, Clara M. Santiveri1, Jorge Santoro1, Montserrat Elías-Arnanz2, S. Padmanabhan2, M. Angeles Jiménez1 1Instituto de Química Física Rocasolano. CSIC. Madrid, Spain. 2Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Spain.

CarD is a global regulator of transcription in Myxococcus xanthus with a two-domain ar-chitecture: a N-terminal domain, CarDNter, that interacts with CarG, and a C-terminal DNA-binding domain resembling eukaryotic high mobility group A proteins [1-2]. CarDNter is the prototype for an entire family of bacterial proteins, described as the CarD-TCRF family [1]. Another member of this family, denoted CdnL (for CarD N-terminus-Like), exists in M. xan-thus. Both CdnL and CarDNter interact with RNA polymerase specifically its β subunit, and despite sequence similarity, the two are not functionally equivalent. In contrast to CarD, CdnL is essential for M. xanthus viability and lacks an intrinsic DNA-binding ability. Nonetheless, CdnL localizes to the nucleoid in vivo, and its depletion results in aberrantly dividing cells. This suggests a role for CdnL in an essential DNA transaction that directly or indirectly affects cell division [2].

To gain further insights into CdnL function and understand its molecular basis, we are exa-mining its structure by NMR. NMR spectra of CdnL showed peak-broadening effects due to a monomer-dimer equilibrium as was also confirmed by analytical ultracentrifugation. By contrast, a stable, monomeric, 110-residue C-terminal domain, CdnL-Cter obtained on limited proteolysis of the 164-residue CdnL yielded NMR spectra of excellent quality. 1H, 13C and 15N NMR signals for CdnL-Cter were completely assigned using standard techniques [3], and its tertiary structure revealed an all α-helical structure composed of five α-helices. Since NMR signals of CdnL-Cter remained unchanged or shifted marginally when in the whole protein, we could analyze NMR data obtained with CdnL. This indicated an all-β N-terminal region in CdnL that is similar to the RNA polymerase interacting domain of transcription coupling repair factors (TRCF). Our structural description of CdnL based on these findings will be presented.

[1] Cayela, M.L., Elías-Arnanz, M., Peñalver-Mellado, M., Padmanabhan, S., and F., Murillo, F.J. (2003) J. Bacteriol. 185, 3527-3537.[2] García-Moreno, D., Abellón-Ruíz, J., García-Heras, F., Murillo, F.J., Padmanabhan, S., and Elías-Arnanz, M. (2010) Nucleic Acid Res..[3] Mirassou, Y., García-Moreno, D., Santiveri, C.M., Santoro, J., Elías-Arnanz, M., Padmanabhan, S., and Jiménez, M.A. (2009) Biomol. NMR Assign. 3, 9-12.

contactauthor:M.angelesJIMénezInstituto de Química Física Rocasolano CSIC. Madrid, Spain.

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Direct Visualization of the oligomerization properties of proteins by in situ atomic Force Microscopy: the Case of the Ferric uptake regulator Fura analyzed under Different redox ConditionsAnabel Lostao1,3, María Luisa Peleato2, Andrés González2, Carlos Gómez-Moreno1,2 and María F. Fillat2 1Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza. Spain. 2Department of Biochemistry and Molecular and Cell Biology and Biocomputation and Com-plex Systems Physics Institute (BiFi), Universidad de Zaragoza. Spain.3Fundación Aragón I+D (ARAID), Aragón, Spain

The knowledge of the oligomerization states of the proteins in different physiological condi-tions can bring significant information about the role they play in the cellular life. The quan-tification of different oligomeric species is often quite difficult to perform. There are useful techniques that can be effective in some cases as gel filtration chromatography, mass analysis or some forms of electrophoresis. However, these techniques do not give good results with many proteins. This work proposes Atomic force microscopy (AFM) as an alternative techni-que to analyze the oligomerization states of proteins. To achieve this purpose immobilization of the protein on a flat substrate in the conditions of study, preferably by adsorption, is re-quired; moreover, choosing an incubation concentration that allows resolving each oligomer individually and obtaining high-resolution images that can distinguish each monomer is nee-ded. In some cases may be necessary to make a volumetric analysis of the features but if the images have a good resolution will be enough to zoom on each feature to determine the type of oligomer. In this work we have used AFM that allows single-molecule imaging to monitor the status of the ferric uptake regulator FurA under different redox conditions mimicking the physiological environment [1]. Fur proteins are global prokaryotic transcriptional regulators. Functional studies of FurA from the cyanobacterium Anabaena sp. PCC 7120 evidenced the influence of the redox environment in the activity of the regulator and its ability to aggregate through disulphide bridges. The estimated FurA average diameter was of 4 nm. In the ab-sence of reducing agents FurA is mainly associated as trimers, being 40º the prevalent angle conformed by the monomers. Reducing conditions induces trimer rearrangement to protein monomers and a major fraction of FurA dimers. Disruption of the dimeric assemblies and appearance of higher order aggregates are induced by oxidation. The homogeneity of the angles exhibited by the trimeric particles, as well as the occurrence of dimers in the presence of DTT, suggests the participation of relatively specific hydrophobic interactions maintaining the dimer. Direct visualization of the regulator under liquid phase at molecular resolution un-ravels the importance of non-polar interactions in FurA dynamics and shows that in Anabaena disulphide bridges are not essential for the dimerization of FurA.

[1] Lostao A., Peleato M.L., Gómez-Moreno C., and Fillat M.F. (2010) BBA: protein and proteomics. In press. DOI: 10.1016/j.bbapap.2010.04.002.

contactauthor:anabellostaoInstituto de Nanociencia de Aragón (INA) Universidad de Zaragoza. Spain.

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Facilitative glucose transporter gLut12: novel data showing electrogenic propertiesAlejandra Pérez1, Jonai Pujol2, Alejandro Reyes1, M. Pilar Lostao2

1Department of Biochemistry, Austral University of Chile, Valdivia, Chile2 Department of Phisiology, Toxicology and Nutrition, University of Navarra, Pamplona 31008, Spain

Background and aims. The facilitative glucose transporters GLUT/SLC2A are integral mem-brane proteins widely distributed in mammalian cells. Until now, 14 GLUT isoforms have been identified, which have been divided into three different classes (I, II and III) based on their sequence comparison [1]. In the present work, we have investigated the transport mechanism of GLUT12 (class III) and shown for the first time electrogenic properties.

Methods. GLUT12 was expressed in Xenopus laevis oocytes and kinetic parameters were obtained by uptake assays with radiolabeled substrates while electrogenic properties were investigated using the two-electrode voltage clamp technique [2].

Results. Electrophysiological experiments showed that glucose and 3-O-methylglucose (5-100 mM) induced inward currents in the presence and absence of sodium in the medium that were of similar magnitude and did not saturate, suggesting that Cl- could be involved in the generation of the currents. Moreover, in the absence of glucose, there was a Na+ leak current. On the other hand, the presence of sodium in the medium increased maximal transport rate of the sugars uptake (Vmax of 984+107 vs. 468+80 pmoles/oocyte/minute in the presence and absence of sodium respectively for glucose) while decreased the affinity (K0.5 of 24.9 mM vs. 7.5 mM in presence and the absence of sodium respectively for glucose). The increase of po-tassium concentration in the medium, from 25 to 100 mM, inhibited both inward current and sugar uptake. Sugars that are not substrates of GLUT transporters, like mannitol and sorbitol, also induced inward current, while genistein, a typical extracellular GLUT inhibitor, inhibited glucose uptake but increased glucose-induced currents.

Conclusion. Sugars induce ion movement trough GLUT12 and Na+ and K+ seem to be invol-ved in the process of transport of glucose, but transport and ion movements are uncoupled, which suggest a substrate-gated ion channel activity for GLUT-12. Experiments in course are conducted to elucidate if Cl- movement is implicated in the sugar-induced currents.

This work was supported by Fundación Marcelino Botín and Ministerio de Ciencia y Tecno-logía (Spanish Government) Grant BFU2007-60420. The Spanish group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF) (Grant BFU2007-30688-E/BFI).

[1] Joost GH and Thorens B (2001) Mol. Membr. Biol. 18, 247- 256 [2] Birnir B, Loo DDF and Wright EM. (1991) Pflugers Archiv-Eur J Physiol. 418, 79-85.

contactauthor:M.pilarlostaoDepartment of Phisiology, Toxicology and Nutrition University of Navarra, Pamplona 31008, Spain

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positioning water molecules around the Mn-Cluster of photosystem ii from pulsed epr experiments.Jesús I. Martínez1, Pablo J. Alonso1, Inmaculada Yruela2, Rafael Picorel2

1Instituto de Ciencia de Materiales de Aragón, ICMA (Universidad de Zaragoza-Consejo Superior de Investigaciones Científicas), Zaragoza, Spain. 2Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Avda. Montañana 1005, Zaragoza, Spain.

Light-driven water oxidation is a very important process in cyanobacteria, green algae and higher plants. It takes place in the oxygen-evolving complex (OEC) within Photosystem II, and it is believed to be catalyzed by a cluster of four manganese and one calcium atoms (Mn-clus-ter) through a five-step cycle (known as Kok cycle). During the Kok cycle two water molecules interact, the Mn-cluster accumulates oxidation equivalents and, finally, molecular oxygen and reduction equivalents are obtained. Mn-cluster structure and function have been studied for decades, but still many questions remain unanswered. Structural data from X-ray diffaction, EXAFS and DFT calculations seem to be difficult to conciliate. The electronic structure in the different Si (i = 0 to 4) states within the Kok cycle are not completely resolved, and the mo-ment and way the interacting water molecules enter the Mn-cluster structure is controversial. Previous studies by means of advanced EPR techniques (1H-ENDOR and 1D ESEEM) resulted to be inconclusive. Here we present 2D ESEEM (HYCORE) measurements of the Mn-cluster in the S2 state that allow us to identify hyperfine interaction with (at least) two protons. Combi-ning the signal resolution of the 2D technique with previously reported ENDOR data, accurate hyperfine constants can be determined. From this, the position of water molecules in the Mn-cluster environment can be discussed. This study shows that 2D ESEEM experiments give relevant information aiming to resolve the puzzle of the Mn-cluster structure and functioning.

contactauthor:JesúsI.MartínezInstituto de Ciencia de Materiales de Aragón, ICMA (Universidad de Zaragoza-Consejo Superior de Investigaciones Científicas) Zaragoza, Spain.

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Conjugated Linoleic acid impairs adipose plasma Membrane permeability and FluidityAna P. Martins1, Paula A. Lopes2, Susana V. Martins2, Ana Madeira1,3, Nuno C. Santos4, José A. M. Prates2, Teresa F. Moura1, Graça Soveral1,5

1REQUIMTE, Dep. Química, FCT-UNL, 2829-516 Caparica, Portugal2CIISA, Faculdade de Medicina Veterinária, TULisbon, Lisboa, Portugal3Institute for Research in Biomedicine (IRB, Barcelona), Spain4Instituto de Medicina Molecular, Faculdade de Medicina-UL, Lisboa, Portugal5Faculdade de Farmácia-UL, Lisboa, Portugal

The relationship between the dietary ingestion of conjugated linoleic acid (CLA), a fatty acid frequently used as a body fat reducing agent, and its presence on cell membranes possible affecting cellular functions is still unexploited. In this study, obese Zucker rats were fed athe-rogenic diets containing distinct saturated fats of vegetable or animal origin (palm oil or ovine fat, respectively), with or without 1% of CLA. Plasma membrane vesicles obtained from visce-ral white adipose tissue were used to assess the effectiveness of dietary fat and CLA isomers membrane incorporation, and its outcome on permeability (stopped flow light scattering) and fluidity (DPH and TMA-DPH fluorescence probes) to water and glycerol. These membrane vesicle preparations were enriched in the adipocyte plasma membrane fraction, as proved by the contents of caveolin, GLUT4, and AQP7. Moreover, they have shown to be osmoti-cally responsive and therefore suitable for transport experiments. The lipid profile analysis of adipose membranes has shown that CLA supplementation did not affect cholesterol and total fatty acids membrane incorporation. Yet, the deposition of cis9,trans11 CLA isomer was higher than the trans10,cis12 isomer. A significant CLA effect on both water and glycerol permeabilities was detected, having dietary groups with CLA lower permeability values. The high activation energy for both water and glycerol transport suggests that permeation occurs mainly through bilayer diffusion. A significant decrease in adipose membrane fluidity was correlated with changes in permeability, which seem to be caused by the incorporation of the trans10,cis12 CLA isomer. Altogether, these results suggest that CLA supplementation through its incorporation in membrane phospholipids has an effect on the fluidity of adipose membranes possibly due to the formation of raft-like micro-domains that promote a decrease in permeability.

contactauthor:anap.MartInsREQUIMTE, Dep. Química, FCT-UNL 2829-516 Caparica, Portugal

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Functional integration of the portal protein of bacteriophage Φ29 in lipid membranes Lara H Moleiro1, Ileana Márquez2, Sonia Moreno3, Jose L Carrascosa3, Iván López-Montero1, Marisela Vélez2 and Francisco Monroy1

1Mechanics of Biological Membranes and Biorheology, Universidad Complutense, 28040 Madrid, Spain 2Instituto de Catálisis y Petroleoquímica, CSIC, 28049 Madrid, Spain 3Centro Nacional de Biotecnología, CNB-CSIC, Campus de Cantoblanco, 28049 Madrid, Spain.

The portal element of bacteriophage viruses constitutes a robust molecular machine for DNA translocation. [1,2] We propose a nanotechnological realization that integrates these power-ful pieces into the lipid membrane of a giant unilamellar vesicle (GUV) on the idea of building a cargo-device useful for gene delivering. In a first stage, as a proof of concept, we consider the efficient incorporation of the hydrophilic protein of the bacteriophage virus Φ29 into a specifically engineered bilayer made of “hydrophylised” lipids. Furthermore, a mutant variant of the protein including a hydrophobic belt (his tags) around will be incorporated to a simpler lipid membrane that contains specific his-binding lipids. Our nano-engineering plan consists of growing GUVs from bilayer membranes built up from proteoliposomes previously prepa-red by extrusion. In our contribution we will present details on the reconstitution methods [3] and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional DNA translocation.

[1] Müller D.J., Engel A., Carrascosa J.L, and Vélez M. (1997) EMBO J. 16, (10) 2547–2553. [2] Méléard P., Bagatolli L.A., and Pott. T. (2009) Methods Enzymol. 465, 161-176. [3] Rigaud JL, Lèvy D (2003). Methods Enzymol. 32, 65-86.

contactauthor:larahMoleIroMechanics of Biological Membranes and Biorheology Universidad Complutense, 28040 Madrid, Spain

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tools-4-Metatool. online suite of web-tools to process stoichiometric network analysis data from MetatooLFederico Morán, Francisco Montero, Sara Vázquez, Daniela Xavier, Alessio Gamba, Paola Bettinelli.

Department of Biochemistry and Molecular Biology I. Universidad Complutense de Madrid. 28040 Madrid.

METATOOL [1] [2] is widely used for stoichiometric analysis (SNA) of metabolic networks. The output of Metatool provides information on the stoichiometric matrix, the convex basis, elementary modes, and enzyme subsets. For systems with large number of reactions, this output is hard to process and visualize, specially for users who have no experience with this software. We have developed suite of web-tools called Tools-4-Metatool (t4m) (http://solea.quim.ucm.es/t4m/) as an online platform that analyses, parses, and manipulates files related with Metatool. It has two major options: Analysis and Compare. Analysis facilitates the visua-lization of the results of the SNA. This option has five tools: (i) MDigraph that draws bipartite directed graphs of Metatool’s metabolic network, its subsets and the subsets’ pathways; (ii) MetaMatrixTXT visualizes the convex basis and elementary modes of Metatool’s output in function of subsets, in vectorial or matrix format; (iii) CBGraphs draws bipartite directed gra-phs of Metatool’s convex basis and its pathways; (iv) EMGraph draws bipartite directed gra-phs of Metatool’s elementary modes and its pathways; and (v) SortEModes orders Metatool’s elementery modes that contain a given metabolite (or a list of metabolites) in a certain side of its reaction. Compare was developed to compare different Metatool’s results from two species, using subsets or elementary modes comparison. It is composed by: (i) Compara tool compares distincts Metatool’s outputs and shows the identical subsets, (ii) ComparaSub com-pares different Metatool’s outputs and shows the first output in function of the subsets of the second, and (iii) ComparaEM compares distincts Metatool’s outputs and shows the identical elementary modes.

The suite t4m also include scripts that generate Metatool’s input based on COBRA [3] SBML files, Cobra2Metatool, or based on a Metatool’s output file that is filtered by a list of convex basis’ enzymes, CBasis2Metatool.

All these tools have been tested with several metabolic networks. In this contribution we will present some examples to illustrate the use and t4m possibilities.

[1] T. Pfeiffer, I. Sanchez-Valdenebro, J. C. Nuno, F. Montero and S. Schuster: METATOOL: For Studying Metabolic Networks, Bioinformatics 15 (1999) 251-257.[2] A. von Kamp and S. Schuster: Metatool 5.0: fast and flexible elementary modes analysis. Bioinforma-tics 22 (15), 2006, 1930-1931.[3] Becker SA, Feist AM, Mo ML, Hannum G, Palsson BO, et al. (2007) Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox. Nat Protoc 2: 727–738.

contactauthor:FedericoMoránDepartment of Biochemistry and Molecular Biology I Universidad Complutense de Madrid. 28040 Madrid e-mail: [email protected]

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a molecular simulation study of the activity of anesthetics molecule on biological membrane of different lipidic composition Sara D. Oliveira Costa1, Rodolfo D. Poraso2 and J. J. López Cascales1 1Universidad Politécnica de Cartagena, Grupo de Bioinformatica y Macromoleculas (BioMac), Área de Química Física, Aulario II, Campus de Alfonso XIII, 30203 Cartagena, Murcia, Spain2Instituto de Matemática Aplicada de San Luis (IMASL)-Dep. de Física, Universidad Nacional de San Luís/CONICET, D5700HHW, San Luís, Argentina.

Local anesthetics prevent both the generation and the conduction of nerve impulse, by bloc-king the influx of ions through the membrane. Most of local anesthetics consist of an aromatic ring and a carbon chain bearing amino groups. We studied benzocaine, an ester-type local anesthetic, for two main reasons. On one hand because it has been one of the most broadly used in topical treatments since its low water solubility. And on the other hand because, un-like other local anesthetics, it is always uncharged at physiological pH.

We focus our interest on the effects of anesthetics on the membrane, with the aim of shed some light on the molecular mechanisms of anesthetics that still remains unclear. With such a goal, a biological membrane was modeled as a lipidic bilayer of DiPalmitoylPhosphatidyl-Choline (DPPC) and DiPalmitoylPhosphatidylSerine (DPPS). Although PC lipids are the major phospholipid constituent of biological membranes, PS lipids are the most important negati-vely charged phospholipid under physiological conditions. The PS lipids play an important role in the cells of the central nervous system, supposedly because of some structural changes associated with the presence of different substances.

In the attempt to elucidate the mechanisms by which the anesthetic interacts with the mem-brane, we used molecular dynamics simulation to study the effects of anesthetic molecule (benzocaine) on biological membrane varying the PS/PC ratio. In this sense, we have determi-ned some dynamic properties of benzocaine molecules, such as translational diffusion coeffi-cient, dipole orientation or the free energy (ΔG) for a benzocaine molecule partitioning from water to the interior of a phospholipid bilayer. Besides, we also studied some physical changes on the membrane by measuring the local pressure in the interior of the lipid bilayers. Our si-mulation results reveals that the presence of DPPS strongly affects dynamic properties of the benzocaine and with the increasing of concentration of DPPS in the membrane we observe a diminution of the barrier of free energy of benzocaine penetration into the membrane. As well, our results evidenced that the presence of the anesthetic in the interior of the bilayer introduces great changes in the pressure profile through the lipid bilayer for certain PS/PC ratios. The simulation results obtained perfectly agree with the experimental correlation between the molecular anesthetic activity and the membrane cell compositions.

Acknowledgments Authors acknowledge the financial support from the Fundación Seneca de la Región de Mur-cia, through the project 08647/PPC/08.

contactauthor:sarad.olIVeIracostaUniversidad Politécnica de Cartagena, Grupo de Bioinformatica y Macromoleculas (BioMac) Área de Química Física, Aulario II Campus de Alfonso XIII, 30203 Cartagena, Murcia, Spain

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Structure of the plakin domain of plectin.Esther Ortega Portero1 , Rubén Martínez Buey1,2 , Arnoud Sonnenberg3, Jose Mª de Pereda1

1Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas – Universidad de Salamanca, Campus Unamuno, 37007. Salamanca. Spain.2Biomolecular Reseach, Structural Biology, The Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.3Netherlands Caner Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Plectin is a member of the plakin family of proteins, which cross-links components of the cytoskeletal system and links these to membrane-associated structures,such as desmosomes and hemidesmosomes.

Plectin is a protein of high molecular weight (500KDa) and exhibits a multi-domain structure. The N-terminal region contains a ~ 1000-residue long sequence conserved between the mem-bers of this protein family, termed the plakin domain.

Based on sequence analysis, the plakin domain of plectin is predicted to be composed of nine Spectrin Repeats (SR) arranged in tandem and a SH3 domain inserted into the central Spectrin Repeat

Each SR consists on three α-helices (A,B and C) packing in a helical bundle with a up-down-up topology. In each pair of tandem SRs, the helix-C of the N-terminal repeat and the helix-A of the C-terminal repeat are fused in a single helix that spans both SR. Therefore, arrays of SRs are expected to form elongated structures.

Here, we present the structure of the central region of the plakin domain of plectin. We have solved the crystal structures of two fragments: SR3-SR4 and SR4-SR5-SH3 to 2.2Å and 2.8Å of resolution, respectively. This is the first structure of an array of SRs with a SH3 domain inserted and shows the disposition of the putative ligand-binding region of the SH3 domain.

In addition, we have obtained structural information at low angle of the region SR3-SR4-SR5-SH3 by SAXS. The SAXS data suggest that this region has an elongated structure (Dmax ~170 Å). The ab initio models built from the SAXS data agree with a pseudo-atomic model built from the crystal structure of the two fragments.

The structure of the plakin domain of plectin, could be a structural model for other plakins and members of the spectrin superfamily.

contactauthor:estherortegaporteroInstituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones CientíficasUniversidad de Salamanca, Campus Unamuno 37007. Salamanca. Spain.

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insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytCInmaculada Pérez-Dorado1,6, Ana González2,3, María Morales2,3, Reyes Sanles1, Waldemar Striker4,6, Waldemar Vollmer4,6, Shahriar Mobashery5, Jose Luis García2, Martín Martínez-Ripoll1, Pedro García2,3, Juan Antonio Hermoso1

1Grupo de Cristalografía Molecular y Biología Estructural, Instituto de Química-Física Rocaso-lano, Consejo Superior de Investigaciones Científicas, Madrid, Spain.2Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.3Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Bunyola, Mallor-ca, Illes Balears, Spain.4Mikrobielle Genetik, Universität Tübingen, Tübingen, Germany.5Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, Indiana, USA.6Present address: Medical Research Council, Laboratoty of Molecular Biology, Cambridge, UK (I.P.-D.), Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dun-dee, Dundee, UK (W.S.) and Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK (W.V.)

The first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimo-dular protein, it is only able to hydrolyze non-cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of non-competent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division.

contactauthor:Inmaculadapérez-doradoGrupo de Cristalografía Molecular y Biología Estructural, Instituto de Química-Física Rocasolano, Con-sejo Superior de Investigaciones Científicas, Madrid, Spain.

81



rotational dynamics of yeast ribosomal stalk proteins in living cells by multiphoton time-resolved Fluorescence polarization micro-spectroscopy Sebastian Raja1, Carolina García1, Miguel A.Sacristán1, Juan P. G. Ballesta2, M.Pilar Lillo1 1Departamento de Química Física Biológica, Instituto de Química-Física “Rocasolano”, CSIC. Serrano 119. 28006 Madrid 2Dinámica y Función del Genoma, CBMSO, CSIC-UAM, Nicolás Cabrera 1, Campus Canto-blanco. 28049 Madrid

This work is an application of the time-resolved fluorescence polarization micro-spectroscopy methods developed in our laboratory, to quantify conformational changes of the yeast riboso-

mal stalk proteins associated to Terpenoid and He-terocyclic Antibiotics interactions. In particular, we are characterizing the flexibility of the C-terminal domain region of the ribosomal stalk proteins in the cytoplasm of Saccharomyces cerevisiae yeast living cells. Biochemical information supports that the pentameric ribosomal stalk protein complex is in-volved in the regulation of protein synthesis, inte-racting with soluble translation factors, and in GTP hydrolysis control during translation. Besides, it has been postulated that the dynamics of the stalk com-ponents has a regulator role in the ribosome function.

This work was supported by Grant CTQ2009-12412

M.C.Wahl and W.Möller (2002) Structure and function of the acidic ribosomal stalk proteins, Curr. Protein Pept. Sci. 3, 93–106

− P.Gonzalo and J.P.Reboud (2003) The puzzling lateral flexible stalk of the ribosome Biol. Cell. 95, 179–193 − C.M.Spahn, M.G.Gomez-Lorenzo, R.A.Grassucci, R.Jørgensen, G.R.Andersen, R.Beckmann, P.A.Penczek, J.P.Ballesta, J.Frank (2004) Domain movements of elongation factor eEF2 and the eukaryotic 80S ribo-some facilitate tRNA translocation. EMBO J. 23, 1008-1019. − R. Francisco-Velilla and M.Remacha (2010) In vivo formation of stable Pentameric (P2α/P1β)-P0-(P1α/P2β) ribosomal stalk complex in Saccharomyces cerevisiae Yeast [Mar 12; pub ahead of print] − C.Santos, M.A.Rodríguez-Gabriel, M.Remacha, J.P.Ballesta (2004) Ribosomal P0 protein domain in-volved in selectivity of antifungal sordarin derivatives. Antimicrob Agents Chemother. 48, 2930-2936.

contactauthor:sebastianraJaDepartamento de Química Física Biológica Instituto de Química-Física “Rocasolano”, CSIC. Serrano 119. 28006 Madrid

Figure: Scheme of the experimental approach.

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Functional characteristics of synaptic vesicles at active ZonesRocío Ruiz1, Raquel Cano1, William J. Betz2 and Lucía Tabares1 1Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, 41009, España.2Department of Physiology and Biophysics, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 (USA)

The fusion of synaptic vesicles with the plasma membrane is a highly regulated process that takes place at specific locations of the presynaptic membrane called Active Zones (AZs). Doc-ked vesicles at AZs belong to the Readily Releasable Pool (RRP). The arrival of an action po-tential at the presynapse triggers the fusion of the vesicles of the RRP. We have studied the kinetics of neurotransmitter release, and the subsequent vesicle recruitment from the Reserve Pool (RP) to AZs, in the neuromuscular junction of mouse in the Levatur Auris Longus (LAL) muscle by intracellular recordings.

The analysis of end-plate potential characteristics shows that the mean number of fused sy-naptic vesicles per action potential (Quantal Content, QC) in these synapses is 40.6 ± 3.92. We have also estimated that the RRP size is 1600 vesicles. Neurotransmitter release during a train of stimuli (100 Hz, 1s) follows a characteristic pattern, initially consisting of a small amount of facilitation followed by an exponential decline in the amount of neurotransmis-sion, corresponding to the consumption of vesicles from the RRP. This phase is followed by a steady-state phase corresponding to the matched balance between vesicle consumption and refilling. The refilling of the RRP takes place by mobilization of new vesicles from the RP and/or by the recycling of vesicles from nearby regions after undergoing a previous round of exocytosis. Under our experimental conditions, and assuming that the RRP depletion is exponential, the time constant of this process is ~ 220 ms, that is, it is necessary 40-50 action potentials to deplete all the vesicles from the RRP. In contrast, the AZs refilling to maintain the plateau during the stimulation train could be fitted with a sigmoid function with a t1/2 of ~ 330 ms. These data are of interest to better understand the dynamic of the vesicle cycle under different stimulation conditions.

contactauthor:rocíoruIzDepartamento de Fisiología Médica y Biofísica Facultad de Medicina Universidad de Sevilla, 41009, España.

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principles of Quantitative evolutionary Design of redox CyclesArmindo Salvador1,2, Pedro Coelho1,2,3, Bharathi Pandurangan1, Michael A. Savageau3

1Molecular Systems Biology Group, Centre for Neuroscience and Cell Biology, and2Chemistry Department, The University of Coimbra, Portugal; 3Department of Biomedical Engineering, University of California - Davis

Do naturally evolved metabolite concentrations and enzyme kinetic parameters adhere to any widespread principles? We addressed this question for one of the most prevalent circuits in metabolic networks: moiety-transfer cycles (MTC), whereby a pair of coupled reactions transfers moiety from a donor (D) to an acceptor molecule (A) via a cycled intermediate (U, moiety-uncharged form, C, charged form). About 70% of the enzyme-catalyzed reactions in Escherichia coli and Saccharomyces cerevisiae participate in MTC. Most MTC play a role analogous to that of power-supply units in electronic circuits. Namely, they must reliably su-pply moiety at the required rate while keeping the concentration of the charged carrier fairly constant. Here, we consider cases where the sum U+C is conserved and both enzymes follow irreversible Michaelis-Menten kinetics, having the respective substrates as the only effectors that participate in the cycle. These conditions are met approximately by redox cycles that feed reducing equivalents to anabolic and antioxidant defense processes. We inferred a set of design principles that must apply so that these MTC perform as effective moiety-supply units. Namely, in the resulting basal steady state [U]/[C] must be low; the supply-side enzyme must satisfy >[U]; the demand-side enzyme must satisfy <[C]. We examined the extent to which concrete biological realizations adhere to these design principles through both detailed studies of well-characterized MTC (1-3) and a broad survey of KMs and metabolite concentra-tions in E. coli and S. cerevisiae. We found that the biological realizations that we analyzed in detail adhere to these design principles. Furthermore, the broad survey indicates that these design principles hold extensively across metabolic processes and organisms.

Acknowledgements

Work supported in part by fellowship SFRH/BD/8304/2002, SFRH/BPD/44905/2008 (FCT-Portugal), and grants PTDC/QUI/70523/2006 (FCT-Portugal) and RO1-GM30054 (US Public Health Service).

References

1. Salvador A & Savageau MA (2003) Proc. Natl. Acad. Sci. USA 100, 14463-14468.2. Salvador A & Savageau MA (2006) Proc. Natl. Acad. Sci. USA 103, 2226-2231.3. Coelho PMBM, Salvador A, & Savageau MA (2009) PLoS Comput Biol 5, e1000319.

contactauthor:armindosalVadorMolecular Systems Biology Group Centre for Neuroscience and Cell Biology

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the role of synaptotagmin 7 in the release of catecholamines from adrenal chromaffin cellsMargarita Segovia1, Anton Maximov2,3, Thomas C. Südhof2,3 & Guillermo Álvarez de Toledo1

1Dpto.Fisiología Médica y Biofísica. Facultad de Medicina. Universidad de Sevilla, 41009 Sevilla, Spain.2Dept. of Cellular & Molecular Physiology, and Howard Hughes Medical Institute, Standford University School of Medicine, Palo Alto CA 94304, USA.3Dept. of Neuroscience, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA.

Synaptotagmins are a family of calcium sensor proteins implicated in hormone and neuro-transmitter release. All Synaptotagmins has two Ca2+-binding domains (C2A y C2B), provi-ding the molecular basis for the calcium sensitivity of release. Synaptotagmin 1 and 7 (Syt 1 y Syt 7) are the most important isoforms in neurons and endocrine tissue since both isoforms account for the total extent of release [1,2]. However, the exact mechanism on how Syt-1 & Syt-7 promote fusion or sculpt the exocytotic fusion pore is not known. We have studied catecholamine release in using Syt7-KO and Syt 7-C2BKI mutant mice [3] by extracellular amperometry and perforated patch clamp recordings. We have observed that catecholamine release is slower at low frequency (0.5 Hz) in Syt 7 WT mice as compare to 15 Hz stimula-tion. These differences remained significant in the mutant C2BKI (Syt7-C2BKI). However, no differences were observed between WT and C2BKI, as shown in the results obtained using conventional Amperometry, where we observed a fast and small release in C2BKI mice. In conclusion, these data indicate differences in the release from chromaffin cells depending on the type of stimulation.

[1].Sudhof, T.C. (2002) J Biol Chem,. 277(10): p. 7629-32.[2].Schonn, J.S. et al. (2008) Proc Natl Acad Sci U S A. 105(10): p. 3998-4003.[3].Maximov, A. et al. (2008) Proc Natl Acad Sci U S A. 105(10): p. 3986-91.[4].Mosharov, E.V. (2008) Methods Mol Biol. 440: p. 315-27.[5].Fulop, T., Radabaugh, S. and Smith, C. ( 2005) J Neurosci. 25(32): p. 7324-32.

contactauthor:MargaritasegoVIaDpto.Fisiología Médica y Biofísica. Facultad de Medicina Universidad de Sevilla 41009 Sevilla, Spain.

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Characterization of the regions involved in the calcium-induced folding of the intrinsically disordered rtX motifs from Bordetella pertussis adenylate cyclase toxin.Ana-Cristina Sotomayor Pérez1, Johanna Karst1, Marilyne Davi1, Iñaki Guijarro2, Daniel La-dant1, Alexandre Chenal1.1Institut Pasteur, CNRS URA 2185, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. 2Institut Pasteur, CNRS URA 2185, Unité de, Département de Biologie Structurale et Chimie, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel β-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is com-posed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain

required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical chan-ges of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved (1). Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a β-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.

(1) Sotomayor Perez, A. C. et al. “Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin.” J Mol Biol 397(2): 534-549.

contactauthor:ana-cristinasotoMayorpérezInstitut Pasteur, CNRS URA 2185, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie28 rue du Dr Roux75724 Paris Cedex 15, France

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anionic Currents in pollen protoplasts from Lilium longiflorumBárbara Tavares1,2,3, Pedro Nuno Dias1,2,3, Teresa Fonseca Moura1, José Alberto Feijó2,3 and Ana Bicho2

1REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universi-dade Nova de Lisboa, 2829-516 Caparica, Portugal.2Instituto Gulbenkian de Ciência, Oeiras, 2780-156 Oeiras, Portugal3Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal, Campo Grande, C2, 1749-016 Lisboa, Portugal

The presence of anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts was studied under symmetrical anionic concentrations by means of the whole-cell configuration of the patch-clamp technique. Three outward-rectifying anionic current popu-lations, ICl1, ICl2 and ICl3, were identified with Cl--based intra and extracellular solutions. These shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for most positive potentials. ICl1 was the current lost by rundown after the establishment of the whole-cell configuration. ICl2 and ICl3 are the currents mea-sured after rundown. ICl2 was insensitive to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) while ICl3 was completely inhibited by 500 μM in the presence of nM [Ca2+]in. Additionally, the effect of three different intracellular calcium concentrations ([Ca2+]in,) in the nM, μM and mM range, was studied showing that all three currents were dependent on the [Ca2+]in. ICl1 increased intensity with increasing [Ca2+]in reaching its maximum with 0.5 mM. ICl2 and ICl3 attained maximum current and conductance values with μM [Ca2+]in. After run-down, the anionic currents measured with NO3--based solutions were indistinguishable from those measured with high Cl- concentrations, indicating that the permeability of the channels for Cl- and NO3- was of the same order of magnitude. This study shows for the first time the presence of a large anion conductance across the membrane of pollen protoplasts, due to the presence of Ca2+-regulated channels. We hypothesize that these channels may be reallocated during the onset of germination to the apical region of the growing pollen tube and, under physiological conditions when the membrane potential is expected to be negative, may be responsible for the large anionic effluxes previously reported by means of self-referencing vibrating probes.

[1] Hamill OP, Marty A, Neher E,Sakmann B and Sigworth FJ. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391:85-100.[2] Holdaway-Clarke TL and Hepler PK. (2003) Control of pollen tube growth: role of ion gradients and fluxes. New Physiologist 159: 539-563 [3] Zonia L, Cordeiro S, Túpy J, Feijó JA. (2002) Oscillatory chloride efflux at pollen tube apex has a role in growth and cell volume regulation and is targeted by Inositol 3,4,5,6-Tetrakisphosphate. The Plant Cell, 14, 2233-2249

contactauthor:BárbarataVaresREQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.

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prediction of solution properties of fully and partially, intrinsically disordered proteinsDiego Amorós, Álvaro Ortega and José García de la Torre

Departamento de Química Física, Facultad de Química, Universidad de Murcia, 30071 Mur-cia, Spain.

A coarse-grained model and simulation procedures are being developed for the prediction of dilute-solution properties of intrinsically disordered proteins. In order to reach long time sca-les, the model is rather simple: it has only one element (bead) per residue. The fully unfolded, flexible segments are represented by a chain of virtual Cα -Cα bonds, with a bending potential and, eventually, a torsional potential, that are parameterized from a data base of protein struc-tures [1,2], along with a hard-spheres excluded-volume interaction. For the folded regions we use the same potentials plus another that accounts for the vicinity interactions that represent and preserve the tertiary structure [2]. Conformational and overall hydrodynamic coefficients properties can be predicted by Monte Carlo procedures [3], and Brownian dynamics can be employed to study the internal dynamics [3]. In a first stage we have parameterized the poten-tials for the flexible regions by fitting our predictions with experimental values [4,5] of the ra-dius of gyration and the hydrodynamic radii of a large set of chemically denaturated proteins. Thus we determine the optimum bead size, and test the excellent quality of the predictions for those properties of denaturated proteins. Next, we are simulating the conformation and dynamics of partially unfolded, intrinsically disordered proteins that contain both structured regions (globular, helical,…) and unstructured parts (flexible linker chains). The preliminary simulation results are qualitative correct: the proteins are able to fold and unfold properly, and in steady conditions the structure remains stable. Quantitative predictions of solution properties, including hydrodynamic coefficients, small-angle x-ray scattering and internal dy-namics/relaxation are being attempted.

Supported by grant CTQ2009-08030 from Ministerio de Ciencia e Innovación and grant 04531/GERM/06 as “Grupo de Excelencia Científica de la Región de Murcia”

[1] G. J. Kleywegt (1997) J. Mol. Biol273, 371-376. [2] V. Tozzini (2005) Curr. Opin. Struc. Biol, 15, 144-150[3] J. García de la Torre, et al (2003) Eur. Biophys. J. 32, 477-486 [4] J.E, Kohn et al (2004) Proc. Natl. Acad. Sci. USA, 101, 12491-12496[5] V.N. Uversky, (2002) Eur. J. Biochem, 269, 2-12

contactauthor:JoségarcíadelatorreDepartamento de Química Física, Facultad de Química Universidad de Murcia 30071 Murcia, Spain.


Posters

91


poster

1/Characterization of novel protease nS3 inhibitors by isothermal titration Calorimetry ...................................................................................................... 106Olga Abian, Adrián Velázquez-Campoy

2/electrokinetics at the nanoscale: Selectivity inversion by multivalent ions in a biological nanochannel ............................................................................ 107Marcel Aguilella-Arzo , Carles Calero, Maria Queralt and Jordi Faraudo

3/Self-assembly of FtsZ in the presence of gtp and Mg involves the concerted formation of a stable oligomeric intermediate: a static light scattering and sedimentation velocity study ........................................................................ 108Ahijado-Guzmán R., Alfonso C., Minton A. P. , Rivas, G.

4/Flip-flop lipid motion, vesicle lysis and reassembly, membrane permeabilization and solubilization: are they interrelated phenomena? . 109Hasna Ahyayauch,Mohammed Bennouna, Alicia Alonso, and Félix M. Goñi

5/Mediators of the intestinal innate immunity inhibit the serotonin transporter activity in intestinal epithelial cells ........................................... 110Carmen Mendoza, Nyurky Matheus, Ruth Iceta, Eva Latorre, José E. Mesonero, Miguel A. Plaza, Pilar. Arruebo, Divina Murillo, Marta Castro, Laura Grasa, Santiago García, Raquel Vicente, Belén Gros and Ana Isabel Alcalde

6/Ceramide fails to displace cholesterol in sphingomyelin-containing model membranes lacking a liquid-disordered phase ............................................. 111Jon V. Busto, Jesús Sot, José Requejo-Isidro, Félix M. Goñi and Alicia Alonso

7/Structure of the third and fourth fibronectin type iii domains of the integrin α6β4 ................................................................................................ 112Noelia Alonso, Rubén M Buey, Héctor Urien, Arnoud Sonnenberg, Jose M. de Pereda1

8/optical monitoring of single synaptic vesicle release from hippocampal neurons in culture ........................................................................................... 113Mª Ángeles Montes Fernández, Imane Jemal and Guillermo Álvarez de Toledo

9/Functional organization of synaptic vesicles in hippocampal neurons monitored by fluorescent probes................................................................... 114Imane Jemal, María-Angeles Montes and Guillermo Alvarez de Toledo

10/establishment of Drosophila melanogaster as a model system to study Multiple acyl Coa Dehydrogenation Deficiency (MaDD) ............................. 115Ema Alves, Barbara J. Henriques, Rui Gonçalo Martinho, Cláudio Moreira Gomes

92

11/a 2DCoS study of the effect of radiation on tgase activity .................... 116N. ANDRAKA, I.DELAARADA, J.GONZALEZ VELASCO, P.BILBAO and J.L.R.ARRONDO1

12/Fusion peptide effects on epitope recognition at Membrane Surfaces by the Broadly neutralizing anti-hiV-1 2F5 Monoclonal antibody ................. 117Nerea Huarte, Aitziber Araujo, Rocío Arranz, José M. Valpuesta and José L. Nieva

13/notable residues for the correct location of flavins in the n-terminal domain of FaD syntethase from C.ammoniagenes ...................................... 118Sonia Susana Arilla Luna, Adrián Velazquez-Campoy and Milagros Medina

14/Lipid bilayer polarity features and thermal behavior in binary mixtures of popC, egg-sphingomyelin and cholesterol ................................................... 119Dalila Arrais, Jorge Martins

15/Molecular dynamics study of h2 diffusion in a [niFeSe]-hydrogenase . 120Carla Baltazar, Cláudio Soares

16/Conformational Stability of the propeptide nh2-terminal of the precursor of pulmonary Surfactant protein B (Sp-B) to ph acidification and Chaotropes. ... 121Ángeles Bañares-Hidalgo, G. López, J. Pérez-Gil and P. Estrada

17/picornavirus 2B viroporin integrates into the er membrane as an α-helical hairpin ............................................................................................ 122Manuel Bañó, Luis Martínez-Gil , Silvia Sánchez-Martinez, José Luis Nieva, Ismael Mingarro

18/Models of coupled molecular motors. ...................................................... 123Sebastián Bouzat and Fernando Falo

19/impact of subdiffusion on the formation and properties of morphogen gradients. a reaction-diffusion approach. .................................................... 124Santos Bravo Yuste, Enrique Abad, and Katja Lindenberg

20/Modeling the folding of gpW and alpha-spectrin Sh3 domain: accurate information from a simple model. ................................................................. 125Pierpaolo Bruscolini, Athi Narayanan Naganathan

21/puzzling out the Mechanical Stability of the Dorsal-Ventral organizer in the Wing imaginal Disc of Drosophila............................................................ 126Oriol Canela Xandri, Francesc Sagués, Jaume Casademunt, and Javier Buceta

22/Molecular dynamics simulations of Dna melting in the presence and absence of some antitumour drugs covalently bonded in the minor groove .................. 127Juan A. Bueren-Calabuig, Carlos M. Galmarini, Carmen Cuevas and Federico Gago1

93


23/aFM Study of the interaction of a synthetic peptide from de gB virus C/ hepatits g virus with the hiV fusion peptide ................................................ 128Òscar Domènech, Josefina Prat, M. Antònia Busquets, M. Asunción Alsina, Victòria Girona

24/regulation of endocytotic fission pores by calcium and dynamin ........ 129José María Cabeza, Jorge Acosta & Eva Alés

25/Membrane topology and protein-protein interactions of pulmonary surfactant protein Sp-B studied by fluorescence spectroscopy .................. 130Elisa J. Cabré, Luís M. S. Loura, Alexander Fedorov, Jesus Perez-Gil, Manuel Prieto

26/pharmacological chaperones for the aromatic amino acid hydroxylases ...131Ana C. Calvo, Tanja Scherer, Angel L. Pey, Ming Ying, Ingeborg Winge, Jeffrey McKinney, Knut Teigen, Jan Haavik, Beat Thöny and Aurora Martinez1

27/global Downhill vs. Barrier-Limited Folding in Single Molecules .......... 132Luis Alberto Campos-Prieto, Jianwei Liu, Ravishankar Ramanathan, Xiang Wang, Douglas English & Victor Muñoz

28/active Zone organization and distribution of synaptic vesicles in the neuromuscular junction of Synaptophysin-phluorin transgenic mice ....... 133Raquel Cano, Rocío Ruiz, Juan José Casañas, William J. Betz and Lucía Tabares

29/protein-structure-based dot plot.............................................................. 134Oscar Conchillo-Sole Xavier Daura

30/adenovirus polypeptide iVa2: looking for a new viral portal ................ 135Menéndez-Conejero, R.; Pérez-Berná, A.J.; Ostapchuk, P.; Hearing, P. ; San Martín, C.

31/Lipid-protein interaction: quantifying protein binding to lipid vesicles using single colour fluorescence correlation spectroscopy (FCS) ................ 136Ana Coutinho, Ana Melo and Manuel Prieto

32/Dynamic interconversion of oligomeric and fibrillar forms of alpha-synuclein ......................................................................................................... 137Nunilo Cremades, Allen Y. Chen, Angel Orte, Massimo Sandal, Paul Dunne, Francesco A. Aprile, Richard Clarke, Carlos W. Bertoncini, Christopher M. Dobson, and David Klenerman.

33/atomic force microscopy of oligomeric arrangements of surfactant protein Sp-B on Langmuir-Blodgett films. .................................................... 138Antonio Cruz, Bárbara Olmeda and Jesús Pérez-Gil

34/aBtS interaction and electron transfer with Cota laccase: a molecular modelling approach ........................................................................................ 139João M. Damas, Cláudio M. Soares

94

35/Characterization of the terminase complex of phage t7 ........................ 140María I. Daudén, Jaime Martín-Benito, Rocio Arranz, Mikel Valle, Juan Carlos Sánchez and Jose L. Carrascosa

36/ph Modulates anionic Currents in pollen protoplasts ............................ 141Pedro Dias, Patricia Domingos, José Feijó, Ana Bicho,.

37/Modulation of a single copper-ligand bond by remote residues determines the stability of plastocyanins. .................................................... 142Estrella Frutos Beltrán, Sofía Díaz Moreno, Jesús Chaboy Nalda, Amin Wagner, Irene Díaz Moreno, Francisco Muñoz López, Simone Raugei, Paolo Carloni, Miguel A. De la Rosa and Antonio Díaz Quintana

38/mrna diffusion explains protein gradients in \textit{Drosophila} early development ................................................................................................... 143Authors: Rui Dilão and Daniele Muraro

39/probing the pore drug binding site of microtubules with fluorescent taxanes: evidence of two binding poses. ...................................................... 144Isabel Barasoain1, Ana M. García-Carril1, Ruth Matesanz, Giorgio Maccari, Chiara Trigili, Mattia Mori, Jing-Zhe Shi, Wei-Shuo Fang, José M. Andreu, Maurizio Botta and J. Fernando Díaz

40/role of hydrogen bonds in peptidic systems from coarse-grained simulations ...................................................................................................... 145Marta Enciso, Antonio Rey

41/a computational study of protein intrafaces and its implication in protein folding .............................................................................................................. 146Vladimir Espinosa Angarica and Javier Sancho

42/Linking Function to Dynamics in globular, Multi-domain and unstructured proteins using nuclear Magnetic resonance Spectroscopy . 148Santiago Esteban-Martín, Robert B. Fenwick, Xavier Salvatella

43/Computational prediction of single thermal stabilizing mutations in proteins of known structure. Method based on electrostatics, polar accessible surface area and disulfide bridging. ............................................ 149Jorge Estrada, Pablo Echenique, Javier Sancho

44/a mesoscopic model for protein-Dna interaction. ................................. 150R. Tapia-Rojo, D. Prada-Gracia, J. J. Mazo, F. Falo.

95


45/Kinetics of the early Stages of transthyretin oligomerization: clues into amyloid formation........................................................................................... 151Zaida L. de Almeida, Tiago Q. Faria, Rui M. M. Brito

46/exploring the folding behavior of peptide dendrimers through computational methods ................................................................................. 152Luís C. S. Filipe, Miguel Machuqueiro, António M. Baptista

47/the specific h-bonding and packing at the interface between theβ-barrel and site 2 are responsible for the different stability of meso- and thermophilic plastocyanins ............................................................................ 153Estrella Frutos-Beltrán, Miguel A. De la Rosa, Antonio Díaz-Quintana

48/the Functional and Structural role of Lys377 in the Secondary transporter Melibiose permease from e. coli ................................................ 154Oliver Fuerst,Yi-Bin Lin, Meritxell Granell, Gérard Leblanc, Víctor A. Lórenz-Fonfría and Esteve Padrós

49/testing the mechanical hypothesis in scaffoldins: the effect of natural linkers and cellulase binding .......................................................................... 156Albert Galera-Prat, Andrés Manuel Vera, Alejandro Valbuena, Javier Oroz & Mariano Carrión-Vázquez

50/Skl and pal: so different but so similar ..................................................... 157Cristina Gallego, José Luis Sáiz, Fernando Díaz, Laura Lagartera, Pedro García, Margarita Menéndez

51/Modulation of Functional properties of Cytochrome c by phosphorylation 158José M. García-Heredia, Maria Salzano, Antonio Díaz-Quintana, Miguel A. De la Rosa, Irene Díaz-Moreno

52/the nMr structure of the n-terminal domain of tubulin Cofactor C reveals a disordered fragment involved in tubulin binding ..................................... 159María Flor García-Mayoral, Raquel Castaño, Juan Carlos Zabala, Manuel Rico, Marta Bruix.

53/eM Characterization of a Clostridium thermocellum Mini-Cellulosome 160Begoña García-Alvarez, Fernando M. V. Dias, José A. M. Prates, Carlos M. G. A. Fontes, Maria João Romão, Ana Luísa Carvalho and Oscar Llorca

54/Folding of prp and the Species Barrier ..................................................... 161Leszek A. Gierusz, David S. Pearson, Michael A. Geeves and Teresa J. T. Pinheiro

96

55/a tailor-made optical tweezers to study the dynamics of the addaB helicase-nuclease ............................................................................................ 162Benjamin Gollnick, Joseph T. P. Yeeles, Mark S. Dillingham and Fernando Moreno-Herrero

56/Characterization of the effect of curcumin on the catalytic activity of pKCα. .............................................................................................................. 163Gómez-Fernández, J.C., Pérez-Lara, A. and Corbalán-García, S.

57/Structure of the n-terminal region of the plakin domain of Bp230 and mapping of the integrin α6β4 binding site ................................................ 164María Gómez Hernández, Arnoud Sonnenberg, José M de Pereda

58/a mechanical model of the tip link suggests its role in acoustic mechanotransduction ..................................................................................... 165Àngel Gómez-Sicilia, Luis Laurence-Martínez, Javier Oroz, Alejandro Valbuena & Mariano Carrión-Vázquez

59/Dielectric constant of planar supported biomembranes measured at the nanoscale ......................................................................................................... 166Laura Fumagalli Aurora Dols Georg Gramse Daniel Esteban Martin Edwards Gabriel Gomila

60/Catalase-like activity of human met-hemoglobin: a mechanistic and kinetic study. .................................................................................................... 167María I. González-Sánchez, Francisco García-Carmona and Edelmira Valero.

61/Direct action in potassium channels of kidney cells by diuretic drugs and aldosterone ..................................................................................................... 168Patrícia Gonçalves, Terese Moura, Ana Isabel Santos, Ana Bicho.

62/implication of the trp domain of trpv1 in functional coupling .............. 169Lucia Gregorio-Teruel, Pierluigi Valente and Antonio Ferrer-Montiel.

63/Biophysics of protein misfolding in metabolic disease: Defective folding and function on etF mutants ......................................................................... 170Bárbara J. Henriques, Rikke K. Olsen, Peter Bross, Cláudio M. Gomes,*

64/Structural rearrangements in the kinase module of FaD synthetase during catalysis ............................................................................................................ 171Beatriz Herguedas, Juan A. Hermoso, Marta Martínez-Júlvez, Milagros Medina

65/reconstitution of bacterial cell division proteins onto phospholipid bilayer nanodiscs ............................................................................................. 172Víctor M. Hérnandez-Rocamora, Germán Rivas

97


66/triptophan mutations in hirsutellin a produce a non-cytotoxic but ribonucleolitically active ribotoxin ................................................................ 173Elías Herrero-Galán, Javier Lacadena, Álvaro Martínez del Pozo, Nieves Olmo, Mercedes Oñaderra and José G. Gavilanes.

67/Single-molecule characterization of the mechanical properties of dsrna by means of optical-tweezers ......................................................................... 174Elías Herrero-Galán, José María Valpuesta, José L. Carrascosa and José Ricardo Arias-González

68/Mechanical Stability of Low-humidity Single Dna Molecules with polycations ....................................................................................................... 175Silvia Hormeño, Fernando Moreno-Herrero, Borja Ibarra, José L. Carrascosa, José María Valpuesta and J. Ricardo Arias-González.

69/Fusion peptide effects on epitope recognition at membrane surfaces by the broadly neutralizing anti-hiV-1 2F5 monoclonal antibody ................. 176Nerea Huarte, Aitziber Araujo, Rocío Arranz, José M. Valpuesta and José L. Nieva

70/the phi29 Dna polymerase presents an active Dna unwinding mechanism ....................................................................................................... 177Borja Ibarra, Jose A. Morín, Ricardo J. Arias-González, José M. Lázaro, Margarita Salas, José M. Valpuesta, José L. Carrascosa

71/exploring the Structural and energetic Determinants of Ligand Binding to transthyretin: Searching guidelines for the rational Design of therapeutic Drugs against ttr amyloidosis ..................................................................... 178Catarina S. H. Jesus Cláudia V. S. Moniz, Pedro Cruz, Adrian Velazquez-Campoy and Rui M. M. Brito

72/reconstitution of e. coli proto-ring complex inside giant unilamelar vesicles made from bacterial inner membranes .......................................... 179Ariadna Martos, Miguel Vicente, Germán Rivas-Caballero* and Mercedes Jiménez*

73/Sphingosine incorporation into negatively-charged lipid bilayers modifies their structural properties .............................................................................. 180Noemi Jiménez- Rojo, Ana R. Viguera, Félix M. Goñi, Alicia Alonso

74/reconstitution of proapoptotic bak function in liposomes reveals a dual role for mitochondrial lipids in the membrane permeabilization process . 181Olatz Landeta, Ane Landajuela and Gorka Basañez

75/Modulation of the FMn electronic properties by the protein environment of anabaena Flavodoxin ................................................................................. 182Isaias Lans, Susana Frago, and Milagros Medina

98

76/When topology is not enough: evaluating a new coarse-grained hybrid potential for protein folding .......................................................................... 183María Larriva* and Antonio Rey

77/exploring the conformational space of pyruvate carboxylase by Cryo-eM, new insights into its mechanism of action .................................................... 184Gorka Lasso, Linda P.C. Yu, David Gil, Song Xiang, Liang Tong, Mikel Valle

78/a revised Model for rnase a oligomerization via 3D Domain Swapping Based on the Biophysical Characterization of its Conformational ensemble in 40% acetic acid ............................................................................................... 185Jorge Pedro López Alonso, Marta Bruix, Josep Font, Marc Ribó, Maria Vilanova, María Ángeles Jiménez, Jorge Santoro, Carlos González, Douglas Laurents.

79/Comparative study of nucleophosmin and nucleoplasmin: two not so similar proteins. ............................................................................................... 186Benoit Lectez, Jorge Falces, Igor Arregi, Sonia Bañuelos, Petr Konarev, Dmitri Svergun, Stefka Taneva and María A. Urbaneja.

80/replacing arg149 by Cys in the melibiose permease hampers one step in the transport cycle .......................................................................................... 187Yi-Bin Lin, Oliver Fuerst, Víctor A. Lórenz-Fonfría, Meritxell Granell, Gérard Leblanc, and Esteve Padrós

81/Cardiolipin, a key component to mimic the e. coli bacterial membrane in model systems membranes ............................................................................ 188Sílvia Lopes, Cristina Neves, Peter Eaton, Paula Gameiro

82/Viscoelastic properties of natural ceramides ........................................... 189Elisa R. Catapano, Iván López-Montero and Francisco Monroy

83/Surfactant inhibition and reactivation as seen in captive bubble surfactometry .................................................................................................. 190Elena Lopez-Rodriguez, Olga L. Ospina, Mercedes Echaide, H.William Taeusch and Jesus Perez-Gil

84/identification of some inhibitors compounds of β - amyloid aggregation. 191Laura Catalina López, José Alberto Carrodeguas, Salvador Ventura, Javier Sancho.

85/Binding and stability of structural mimics of the phosphorylated species of the histidine phosphocarrier proteins of Streptomyces coelicolor. ........ 192Miriam López-Pérez, Ana-Isabel Martínez-Gómez, David Aguado-Llera, Sergio Martínez-Rodríguez, Javier Gómez and José L. Neira

99


86/Structural insights into the activation mechanism of melibiose permease by sodium binding .......................................................................................... 193Meritxell Granell, Xavier León, Gérard Leblanc, Esteve Padrós and Víctor A. Lórenz-Fonfría.

87/the behavior of pyrene in popC and popC/cholesterol bilayers: a molecular dynamics study .............................................................................. 194Luís Loura, António do Canto

88/a molecular dynamics study of ligand imprinting .................................. 195Diana Lousa; António M. Baptista; Cláudio M. Soares

89/Membrane pore formation by the human immunodeficiency virus type-1 neutralizing anti-gp41 antibody 2f5 .............................................................. 196Rubén Maeso‡, Renate Kunert§, and José L. Nieva‡

90/Constant-ph molecular dynamics simulation with explicit membranes: study of peptide-bilayer interactions ............................................................ 197P. R. Magalhães, M. Machuqueiro, A.M. Baptista

91/Steady-state fluorescence quenching with pyrenyl probes in fluid bilayers examined by a two-dimensional kinetic formalism ..................................... 198Miguel Manuel, Jorge Martins

92/Dependence of mean evolutionary time with the mutation rate .......... 199Arturo Marín, Hector Tejero, Juan Carlos Nuño and Francisco Montero.

93/Molecular Dynamics analysis of the conformational change of paramyxovirus F protein in the initial steps of membrane fusion. ............. 200Fernando Martín-García, Jesús I. Mendieta-Moreno , Jesús Mendieta and Paulino Gómez-Puertas.

94/Spectroscopic Studies on the interaction of the cationic conjugated polyelectrolyte poly{[9,9-bis(6’-n,n,n-trimethylammonium) hexyl] fluorene-phenylene} bromide (htMa-pFp) with human Serum albumin. ................ 201Maria José Martínez-Tomé, Rocío Esquembre, Ricardo Mallavia and C. Reyes Mateo. .

95/thermodynamics of chlorpromazine association with lipid bilayers .... 202Patrícia A. T. Martins, Adrián Velazquez-Campoy, Winchil L. C. Vaz and Maria J. Moreno

96/Structural basis of the FMn midpoint reduction potential modulation in anabaena Flavodoxin ..................................................................................... 203Marta Martínez-Júlvez, Beatriz Herguedas and Milagros Medina.

100

97/internalization and releasing of cholesterol in the endosome: stability and importance of the fourth and fifth binding module of the LDL receptor. .......... 204Juan E. Martínez, Xabier Arias-Moreno, Adrián Velazquez-Campoy and Javier Sancho

98/Molecular recognition of azathilones by microtubules. ......................... 205Ruth Matesanz, Fabian Feyen, Karl-Heinz Altmann and J. Fernando Díaz.

99/the role of gln 61 in ras p21 gtp hydrolysis: a QM/MM study .............. 206Fernando Martin-Garcia, Paulino Gomez-Puertas and Jesus Mendieta

100/nanostructuration of Ferritin nanoparticles on Sensors using Dip-pen nanolithography ............................................................................................. 207Rocío de Miguel, Elena Bellido, María José Martínez-Pérez, Daniel Maspoch, Javier Sesé, Carlos Gómez-Moreno, Fernando Luis, Daniel Ruiz-Molina, Anabel Lostao

101/reconstitution of bacterial cell division protein complexes in membrane-coated micro-beads: fluorescence spectroscopy studies. ....... 208Begoña Monterroso, Ariadna Martos, Rubén Ahijado, Carlos Alfonso, Mercedes Jiménez, Germán Rivas, Silvia Zorrilla

102/partition of amphiphilic molecules to lipid bilayers by itC ................. 209Maria João Moreno, Margarida Bastos and Adrian Velazquez-Campoy

103/the transcriptional regulatory network of Mycobacterium tuberculosis .210Joaquín Sanz, Jorge Navarro, Ainhoa Arbués, Carlos Martín, Pedro Marijuán, Yamir Moreno.

104/Kinetic Modeling of Single Molecule Data: the phi29 Dna polymerase unwinding activity. ......................................................................................... 211José A. Morín , Borja Ibarra , J. Ricardo Arias-González , Margarita Salas, José M. Valpuesta, José L. Carrascosa.

105/energetics of nucleotide-induced DnaK conformational states. ......... 212Fernando Moro, Stefka G. Taneva, Adrián Velázquez-Campoy and Arturo Muga

106/Snapin is an oligomeric natively-unfolded protein, which forms a “fuzzy”, non-structured complex with snap-25 ........................................... 213Aaron Navarro, José A. Encinar, Jesús Prieto, Javie Gómez, Alfonso Martínez-Cruz, Gregorio Fernández-Ballester, José L. Neira and Antonio Ferrer-Montiel

107/Quantification of Synaptic Vesicles at the neuromuscular Junctions in Spinal Muscular atrophy Mice ........................................................................ 214Margret Neher, Rocío Ruiz and Lucía Tabares

101


108/the nK1 receptor: Strategies of expression, purification and refolding. . 215Mikhail Orel, Esteve Padrós, Joan Manyosa

109/Modelling Spontaneous activity in neuronal Cultures......................... 216Javier G. Orlandi, Enric Álvarez-Lacalle, Núria Amigó, Jordi Soriano1 and Jaume Casademunt.

110/nanomechanics of the tip-link cadherins and its role in hearing ........ 217Javier Oroz, Alejandro Valbuena, Albert Galera-Prat, Rubén Hervás, Ulrich Müller, & Mariano Carrión Vázquez

111/Coupling lipid and protein composition to optimize surface activity of synthetic and semi-synthetic pulmonary surfactant preparations ............. 218Olga L. Ospina, David Schürch, Antonio Cruz and Jesús Pérez-Gil

112/effect of hydrophobic surfactant proteins Sp-B and Sp-C on permeability of phospholipid membranes .......................................................................... 219Elisa Parra, Lara H. Moleiro, Antonio Alcaraz, Iván López-Montero, Antonio Cruz, V.M. Aguilella, Francisco Monroy, Jesús Pérez-Gil

113/effect of crowding by Dextrans on the hydrolysis of n-succinyl-L-phenyl-ala-p-nitroanilide catalyzed by alpha-chymotrypsin ................................... 220I. Pastor, E. Vilaseca, S. Madurga, J. L. Garcés, M. Cascante and F. Mas

114/Structural studies in mrna packaging machinery ................................. 221Pena A, Gewartowski K, Montoya G, Dziembowski A and Valpuesta JM.

115/Molecular recognition of peloruside a by microtubules. the C24 primary alcohol is essential for biological activity ...................................................... 223Benet Pera, Mina Razzak, Chiara Trigili, Oriol Pineda, Angeles Canales, Rubén M. Buey, Jesús Jiménez-Barbero, Peter T. Northcote, Ian Paterson, Isabel Barasoain and José Fernando Díaz

116/Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomes ................................................................................. 224José M de Pereda, Pilar Lillo, Arnoud Sonnenberg

117/Modelling the F1-atpase ........................................................................ 225Rubén Pérez

118/Biophysical studies reveal weak points in the adenovirus capsid ....... 226Pérez-Berná AJ, Menéndez-Conejero R, Menéndez M, Flint SJ and San Martín C.

119/the three-dimensional characterization of the DnaK:grpe complex .. 227María Angeles Pérez-Calvo, Judit Perales, Fernando Moro, Oscar Llorca, Arturo Muga and José María Valpuesta

102

120/the Cp43´ complex of the cyanobacterium Synechocystis pCC 6803 under Fe-starvation. ........................................................................................ 228R. Picorel, M.A. Luján, P. Llorente, R. Cases, M. Seibert and R. Jankowiak.

121/physiological splice variant of hMgCS2 gene ....................................... 229Mónica Ramos, Eduardo López-Viñas, Beatriz Puisac, María Arnedo, Sebastián Menao, María Concepción Gil-Rodríguez, María Esperanza Teresa-Rodrigo, Angeles Pié, Feliciano J. Ramos, Paulino Gómez-Puertas, Juan Pié.

122/three dimensional structure model of the nipBL heat repeats .......... 230María Concepción Gil-Rodríguez, Eduardo López-Viñas, Beatriz Puisac, María Arnedo, María Esperanza Teresa-Rodrigo, Milagros Ciero, Angeles Pié, Feliciano J. Ramos, Paulino Gómez-Puertas, Juan Pié.

123/Structural characterization of protein-protein complexes by integrating computational docking with small-angle scattering data ........................... 231Carles Pons Marco D’Abramo Dmitri I. Svergun, Modesto Orozco,Pau Bernadó, Juan Fernández-Recio

124/probing the interaction between divalent cations and ompF channel residues: inversion of ionic selectivity and pKa shift of acidic residues ...... 232María Queralt-Martín, Elena García-Giménez, M. Lidón López, Antonio Alcaraz

125/Salt-dependent ph sensing and cooperative transport in a protein ion channel revealed by current noise, conductance and selectivity experiments 233María Queralt-Martín, Elena García-Giménez, Vicente M. Aguilella, Antonio Alcaraz

126/nucleoplasmin binds histone h2a-h2b dimers through its distal face 234Isbaal Ramos, Jaime Martín-Benito, Ron Finn , Laura Bretaña, Kerman Aloria, Jesús M Arizmendi, Juan Ausió, Arturo Muga, José M Valpuesta and Adelina Prado.

127/Metallic cations modulate the nanomechanical response of phospholipid model membranes. a force spectroscopy study. ................... 235L. Redondo-Morata , G. Oncins and F.Sanz

128/immobilization and characterization of Kcsa potassium channel into sol-gel glasses ........................................................................................................ 236Rocío Esquembre, María L. Renart, José A. Poveda, José M. González-Ros and C.Reyes Mateo.

129/iL-1β effect on gLut5 intestinal transporter ........................................ 237Alberto García-Barrios , Natalia Guillén , Sonia Gascón , Jesús de la Osada , Isabel Escudero, Carmen Viñuales, Mª Carmen Rodríguez-Yoldi, Mª Jesús Rodríguez-Yoldi .

103


130/Structural Characterization of the Centrosomal protein na14 ............ 238Rodríguez-Rodríguez, M., Treviño, M., Laurents, D.V., Arranz, R., Rico, M., Bruix, M. and Jiménez, M.A.

131/Modulation of the activity of different variants of Bone Morphogenetic protein-2. ......................................................................................................... 239Romero-Romero ML, López-Lacomba JL, Sanchez-Ruiz JM

132/role of charge neutralization in the folding of the carboxy-terminal domain of histone h1. ..................................................................................... 240Alicia Roque, Núria Teruel, Inma Ponte, Pedro Suau.

133/analysis in response to osmotic and heat stress in ustilago maydis. .. 241Karina Salmerón-Santiago, Juan Pablo Pardo-Vázquez, Oscar Flores-Herrera and Guadalupe Guerra-Sánchez*

134/involvement of ferredoxin-naDp+ reductase FaD and naDp+/h binding domains for optimal catalytic complex formation and hydride transfer efficiency. ......................................................................................................... 242Ana Sánchez-Azqueta, Matías Musumeci, Eduardo Ceccarelli and Milagros Medina

135/interaction of nucleoside derivatives with the human concentrative nucleoside transporters hCnt1, hCnt2 and hCnt3 using electrophysiological techniques. ...................................................................................................... 243Carlos Sancho-Mateo, Edurne Gorraitz, Carmen Sanmartín, Itziar Pinilla-Macua, Marçal Pastor-Anglada, Mª Pilar Lostao

136/atomic force microscopy-based molecular recognition of fibrinogen receptor on human erythrocytes ................................................................... 244Filomena A. Carvalho, Simon Connell, Gabriel Miltenberger-Miltenyi, Sónia Vale Pereira, Alice Tavares, Robert A.S. Ariëns, Nuno C. Santos

137/Spreading of persistent infections in heterogeneous populations ..... 245Joaquín Sanz, Luis Mario Floría, Yamir Moreno.

138/Function of the trwB transmembrane domain in the protein-protein interaction and cellular location .................................................................... 246Rosa .L. Segura, Sandra Águila, Ana J. Vecino, Begoña Ugarte, Fernando de la Cruz, Félix M. Goñi, Itziar Alkorta

139/nMr structural studies on peptides mimicking the pre-membrane stem region of the hiV-1 gp41 protein ................................................................... 247Soraya Serrano, Nerea Huarte, Beatriz G. de la Torre, David Andreu, José L. Nieva, M. Angeles Jiménez.

104

140/Characterization of the putative residues involved in the synthesis of FMn and FaD in FaD synthetase from Corynebacterium ammoniagenes .. 248Ana Serrano, Adrián Velázquez-Campoy and Milagros Medina.

141/new biophysical approaches using in the environmental situation ... 249Shamiyan A.G.

142/Cardiac dynamics : Spiral drift and meandering in heterogeneous heart tissues ............................................................................................................... 250J. Bragard and A. Simic

143/Modifying the flexibility of bacteriorhodopsin helices impairs proton transport efficiency ......................................................................................... 251Rosana Simón-Vázquez, Tzvetana Lazarova, Alejandro Perálvarez-Marín, José Luís Bourdelande, Esteve Padrós

144/approaching Fluoquinolone Drug Delivery Systems ............................ 253Isabel Sousa, Paula Gameiro

145/effect of chitosan degradation on its interaction with beta-lactoglobulin 254Hiléia K. S. Souza, Maria do Pilar Gonçalves and Javier Gómez

146/Function and regulation of Vitis vinifera aquaporins heterologously expressed on yeast .......................................................................................... 255Luís Leitao, Ana P. Martins, Ana Madeira, Catarina Prista, Maria C. Loureiro-Dias, Teresa Moura, Graça Soveral

147/Conjugated Linoleic acid impairs adipose plasma Membrane permeability and Fluidity ............................................................................... 256Ana P. Martins, Paula A. Lopes, Susana V. Martins, Ana Madeira, Nuno C. Santos, José A. M. Prates, Teresa F. Moura, Graça Soveral

148/understanding lactose permease lipid selectivity from aFM observations of supported planar bilayers .......................................................................... 257Carme Suárez-Germà, Oscar Domènech, Laura Picas, Jordi Hernández-Borrella, M. Teresa Montero

149/Studying of therapeutic activity of an artemisia absinthium `s extract at level of bilayer lipid membranes .................................................................... 258Sukiasyan A.R., Hambardzumyan A.F., Tadevosyan A.V.

150/Structure of molten globule by equilibrium ф-analysis: helicobacter pylori apoFLaVoDoXin ............................................................................... 259Renzo Torreblanca Gonzáles ; Javier Sancho

105


151/Synaptic vesicle expression of vaCht-phluorin in septal neurons ...... 260Laura Torres-Benito, Rocío Ruiz, M. Ángeles Montes, Guillermo Álvarez de Toledo, Lucía Tabares.

152/Sar of hybrids of the microtubule stabilizing natural products dictyostatin and discodermolide. investigations into the relationship between binding affinity and cellular cytotoxicity. ...................................... 261Chiara Trigili, Guy J. Naylor, Nicola M. Gardner, Ian Paterson, J. Fernando Díaz and Isabel Barasoain1

153/hamiltonian replica-exchange methods for protein-structure refinement including experimentally determined distance restraints .......................... 263Dunja Urosev, Roman Affentranger, Xavier Daura

154/Mechanical properties of β-catenin ...................................................... 264Alejandro Valbuena, Andrés Manuel Vera, Javier Oroz, Margarita Menéndez & Mariano Carrión-Vázquez

155/Discrete and continuous hybrid petri nets to analyze the dynamic behaviour of the h2o2-detoxifying pathway in chloroplasts ..................... 265Edelmira Valero, Hermenegilda Macià, Mª Isabel González and Valentín Valero

156/reconstitution into liposomes enhances nucleotide binding affinity of trwB conjugative coupling protein ............................................................... 266Ana Julia Vecino, Rosa de Lima Segura, Begoña Ugarte-Uribe, Sandra Águila, Fernando de la Cruz, Félix M. Goñi, and Itziar Alkorta

157/Conformational Landscape of hepatitis C nS3 protease ...................... 267Olga Abian, Sonia Vega, Jose Luis Neira, Adrian Velazquez-Campoy

106

poster 1

Characterization of novel protease nS3 inhibitors by isothermal titration CalorimetryOlga Abian1,2,3, Adrián Velázquez-Campoy1,4

1Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zarago-za, 50018 Zaragoza, Spain.2Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), ISCIII.3Aragon Health Sciences Institute (I+CS), Zaragoza, Spain.4Fundación ARAID, Diputación General de Aragón, Spain.

Approximately 170 million people worldwide are chronically infected with the hepatitis C virus (HCV). The current combination therapy is not effective. New drugs targeting HCV en-coded enzymes are being evaluated in clinical trials and have shown viral load reductions. The majority of drug candidates target either the protease activity of the non-structural protein NS3 or the viral polymerase NS5B. NS3 is a bifunctional protein containing both protease and helicase domains. The protease activity of the hepatitis C virus non-structural protein 3 (NS3) is essential for viral replication. The present work used NS3 protease as target molecule. The aim of this study was to test the inhibitory capacity of a thousand of compounds library and study their binding mechanism.

High-throughput screening of a library of thousand of chemical compounds was performed. Some of them were chosen as lead compounds and their inhibition constants were measured using a FRET (Fluorescence Resonance Energy Transfer) substrate in a multiplate fluorimeter. Affinity constant and binding enthalpy was measured by ITC (Isothermal Titration Calorime-try). This technique is especially appropriate for obtaining direct information on the energe-tics of enzyme-inhibitor interaction.

Some of the lead compounds showed promising inhibition constant values (µM and sub-µM order). Comparison with their affinity constants determined by ITC allowed discriminating the type of inhibition. The presence of NS4A peptide cofactor influence the inhibition constant of some compounds, suggesting a cooperative binding interaction. ITC results revealed that some compounds exhibited favourable enthalpy values, thus, constituting excellent candida-tes for further optimization.

As result of this study, some compounds have been identified as good candidates to be opti-mized as potential drugs against Hepatitis C Virus. We know that some compounds showed an enthalpically driven binding to NS3 protease, and, as it has been previously reported, this type of binding will allow us to have a good starting point to improve their binding affinity.

contactauthor:olgaaBIanInstitute of Biocomputation and Physics of Complex Systems (BIFI) Universidad de Zaragoza, 50018 Zaragoza, Spain.

107


poster 2

electrokinetics at the nanoscale: Selectivity inversion by multivalent ions in a biological nanochannelMarcel Aguilella-Arzo1 , Carles Calero2, Maria Queralt1 and Jordi Faraudo2

1Biophysics Group, Department of Physics, Universitat Jaume I, 12080 Castelló, Spain.2Institut de Ciència de Materials de Barcelona (CSIC), Campus de la UAB, E-08193 Bellaterra, Spain

Email: [email protected]

The role of multivalent counterions in electrokinetic phenomena has attracted a great deal of theoretical and experimental interest [1-3] due to their ability to induce complex and rich new phenomena. One new example has been recently added, namely the observation that a cer-tain biological nanochannel (the OmpF bacterial porin) reverses its selectivity in presence of multivalent cations [4]. The OmpF is a relatively wide ionic channel in the sense that it allows the simultaneous permeation of both cations and anions (in hydrated form) at high rates. In presence of monovalent electrolyte, it has a slight cationic selectivity (i.e. the flux of cations is larger than the flux of anions across the pore) which is well understood from basic elec-trostatic concepts (the pore has overall negative charge at pH=7). However, new experimental results show that the cationic selectivity of the channel found for 1:1 electrolytes is reversed for sufficiently high concentrations of 2:1 or 3:1 electrolytes.

In this work, we discuss the results of new high-performance all-atom molecular dynamics simulations of the ionic transport across this biological nanochannel (with structure obtained from X-ray experiments) in the presence of electrolyte. We study the transport of monovalent, divalent and mixtures of monovalent and divalent ions across the channel. The simulations allow us to identify the microscopic mechanism responsible for selectivity inversion, demons-trating

the prominent role of electrostatic correlations (leading to cation binding) between multiva-lent cations and negatively charged groups present at the pore walls. Such correlations were previously shown to be relevant to account for charge reversal in biological and biomimetic systems such as phospholipid bilayers and liposomes [3].

[1] A.Y. Grosberg et al., Rev. Mod. Phys. 74, 329 (2002); Y. Levin, Rep. Prog. Phys. 65 1577 (2002); H, Boroudjerdi et al., Phys. Rep. 416 129 (2005)[2] J. Faraudo and A. Travesset, J. Phys. Chem. C 111, 987 (2007); J. Faraudo and A. Travesset, Biophys. J. 92, 2806 (2007)[3] J. Lyklema, Adv. Colloid Interface Sci. 147, 205 (2009); A. Martín-Molina et al., J. Phys. Condens. Matter 15, S3475 (2003); F. H. van der Heyden et al., Phys. Rev. Lett. 96, 224502 (2006)[4] A. Alcaraz et al., Biophys. J. 96, 56 (2009)

contactauthor:MarcelaguIlella-arzoBiophysics Group, Department of Physics Universitat Jaume I, 12080 Castelló, Spain.

108

poster 3

Self-assembly of FtsZ in the presence of gtp and Mg involves the concerted formation of a stable oligomeric intermediate: a static light scattering and sedimentation velocity study Ahijado-Guzmán R.1, Alfonso C.1, Minton A. P. 2, Rivas, G.1

1Departamento de Biología Físico-Química, Centro de Investigaciones Biológicas, CSIC, Rami-ro de Maeztu 9, 28040 Madrid, E-mail: [email protected] of Biochemistry and Genetics, NIDDK, NIH, Bethesda, MD

The GTP-dependent assembly of the FtsZ protein (bacterial ancestor of the eukaryotic tubu-lin) is an important reaction for the formation of dynamic septal ring during cell division. In this work, the assembly of FtsZ in the presence of constantly replenished GTP was studied as a function of Mg concentration at neutral pH under steady-state conditions by sedimentation velocity and concentration-gradient light scattering. Sedimentation velocity measurements confirmed previous measurements indicating cooperative appearance of a narrow size dis-tribution of stable intermediates with increasing concentration, the size of which increased with increasing Mg concentration. The concentration dependence of light scattering indepen-dently verifies the cooperative appearance of a narrow size distribution of oligomeric species, and in addition provides an estimate of the average size of these species, which corresponds to 35-40 FtsZ protomers at millimolar Mg concentration. We have also observed that the degree of assembly (according to the scattering signal) is reduced upon decreasing the salt concentration from 0.5 to 0.05 M potassium, which is opposite to the effect previously des-cribed on GDP-bound FtsZ oligomer formation.

contactauthor:ahIjado-guzMánrDepartamento de Biología Físico-Química, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid E-mail: [email protected].

109


poster 4

Flip-flop lipid motion, vesicle lysis and reassembly, membrane permeabilization and solubilization: are they interrelated phenomena?Hasna Ahyayauch1,Mohammed Bennouna2, Alicia Alonso1, and Félix M. Goñi1

1Unidad de Biofısica (Centro Mixto CSIC-UPV/EHU) and Departamento de Bioquimica, Uni-versidad del Pais Vasco, Aptdo. 644, 48080 Bilbao, Spain.2Department of Biology, Faculty of Sciences Semlalia,Marrakech, Morocco

Surface active amphiphilic compounds known as surfactants find interesting applications in Biology. Because of their amphiphilic nature, surfactants partition into biological membranes, altering their physicochemical properties even at concentrations below those causing mem-brane solubilization (i.e. in the so-called stage I of detergent-membrane interaction). The main subsolubilizing detergent effects on membranes are transmembrane lipid motion (flip-flop), breakdown of the membrane permeability barrier (leakage), and vesicle lysis/reassembly. The main question behind our study was whether detergent induced flip-flop motion, bilayer per-meabilization, and bilayer lysis/reassembly could be related to each other by a cause-effect relationship (i.e., whether they could be described as different consecutive steps, necessarily correlated within the framework of a larger event such as bilayer solubilization). Our reply to this question is negative, the above events appear to occur independently from each other, and not as successive steps in a single overall event.

contactauthor:hasnaahyayauchUnidad de Biofısica (Centro Mixto CSIC-UPV/EHU)

110

poster 5

Mediators of the intestinal innate immunity inhibit the serotonin transporter activity in intestinal epithelial cellsCarmen Mendoza1, Nyurky Matheus1, Ruth Iceta1, Eva Latorre1, José E. Mesonero1, Miguel A. Plaza1, Pilar. Arruebo1, Divina Murillo1, Marta Castro1, Laura Grasa1, Santiago García2, Raquel Vicente2, Belén Gros2 and Ana Isabel Alcalde1

1Physiology. Department of Pharmacology and Physiology. Faculty of Veterinary Sciences. Zaragoza. Spain. 2Hospital Miguel Servet. Zaragoza. Spain

Serotonin transporter (SERT) expressed in the intestinal epithelium determines 5-HT availa-bility and activity. SERT activity has been described to be altered in chronic intestinal patho-logies such as inflammatory diseases. Toll-like receptors are pattern recognition receptors (PRRs) that play a central role in the initiation of innate cellular immune reponses. Intestinal epithelial cells have been shown to express these receptors and their activation has been described to be tightly and coordinately regulated. Most of the studies about TLRs has de-termined their involvement in immunological responses, however their role in the epithelial physiology mediated by 5-HT can not be discarded. A previous study has demonstrated that TLR4 activation by LPS affects SERT activity and expression [1], however the effect of TLR3, which recognizes double-stranded RNA (dsRNA), on SERT remains unknown. Therefore, the aim of the present work was to determine whether TLR3 activation affects SERT activity and, if so, to determine the mechanisms involved.

The human enterocyte-like cell line Caco-2, which express endogenously SERT, has been used to carry out this work. The results show that these cells express TLR3. When the cells were treated with Poly (I:C) (TLR3 ligand), SERT activity diminished. Apical-basal and basal-apical 5-HT fluxes were also reduced by Poly (I:C) treatment. The activation of TLR3 did not affect the expression level of SERT mRNA and protein. The inhibition of SERT activity yielded by TLR3 seemed to involve the p38 MAPK pathway, however cAMP, PKC or JNK did not seem to be implicated. Since TLR3 activation decrease SERT activity and consequently increase 5-HT extracellular availability, the 5-HT vs TLR3 feed-back was determined and the results have shown that Caco-2 treatment with 5-HT did not affect TLR3 protein expression levels in these cells.

Previous studies carried out in our laboratory had concluded that several factors involved in intestinal inflammation affected SERT activity and expression [1-3]. The present work corro-borates this conclusion and indicates that TLR3 may be also involved in the alteration of the intestinal serotoninergic system mediated by SERT.

[1] Mendoza C, Matheus N, Iceta R, Mesonero JE and Alcalde AI. (2009) Innate Immun. 15, 243-250. [2] Matheus N, Mendoza C, Iceta R, Mesonero JE and Alcalde AI. (2009). Biochem Pharmacol 78, 1198-1204.[3] Matheus N, Mendoza C, Iceta R, Mesonero J and Alcalde AI. (2010) J Pineal Res 48, 332-339.

contactauthor:anaIsabelalcaldePhysiology. Department of Pharmacology and Physiology. Faculty of Veterinary Sciences. Zaragoza. Spain.

111


poster 6

Ceramide fails to displace cholesterol in sphingomyelin-containing model membranes lacking a liquid-disordered phaseJon V. Busto1,2, Jesús Sot1,2, José Requejo-Isidro1, Félix M. Goñi1,2 and Alicia Alonso1,2

1Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU)2Departamento de Bio-química y Biología Molecular, Universidad del País Vasco, Bilbao, Spain

Cell membranes rich in cholesterol (Chol) e.g. plasma membranes, may well be lacking liquid-disordered domains. In order to study the Interactions of sphingomyelin and ceramide un-der those conditions, a set of different biophysical approaches has been used to explore the phase behaviour of palmitoylsphingomyelin (pSM)/Cholesterol (Chol) model membranes in the presence and absence of palmitoylceramide (pCer). Fluorescence spectroscopy of di-4-ANEPPDHQ-stained pSM/Chol vesicles and atomic force microscopy of supported planar bila-yers show gel (Lβ)/liquid-ordered (Lo) phase coexistence within the range XChol = 0-0.25, at 22ºC. At the latter compositional point and beyond, a single liquid-ordered pSM/Chol phase is detected. In ternary pSM/Chol/pCer mixtures, differential scanning calorimetry of multi-lamellar vesicles and confocal fluorescence microscopy of giant unilamellar vesicles concur in showing immiscibility, but no displacement, between liquid-ordered cholesterol-enriched (pSM/Chol) and gel-like ceramide-enriched (pSM/pCer) phases at high pSM/(Chol+pCer) ra-tio. At higher cholesterol contents, pCer is unable to displace cholesterol at any extent, even at XChol < 0.25. Interestingly, an opposite strong cholesterol-mediated pCer displacement from its tight packing with pSM is clearly detected, completely abolishing the pCer ability for the generation of large microdomains and giving rise instead to a single ternary phase.

contactauthor:aliciaalonsoUnidad de Biofísica (Centro Mixto CSIC-UPV/EHU)

112

poster 7

Structure of the third and fourth fibronectin type iii domains of the integrin α6β4Noelia Alonso1, Rubén M Buey1,2, Héctor Urien1, Arnoud Sonnenberg3, Jose M. de Pereda11Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas – Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain.2Biomolecular Research, Structural Biology, the Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.3Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

The integrin α6β4 is a receptor for laminins, which provides stable adhesion of epithelial cells to the basement membranes [1]. In addition, α6β4 promotes keratinocyte migration during wound healing and favours invasion of carcinomas into the surrounding tissue [2, 3]. This integrin is a component of the hemidesmosomes (HD), protein complexes that mediates the stable anchoring of basal epithelial cells to the basement membrane [4].

The cytoplasmic domain of the β4 subunit is responsible for most of the intracellular inte-ractions of α6β4, including the interaction with other hemidesmosomal components such as plectin and BPAG1e [5]. This cytoplasmic region of β4 contains a Calx-β domain and four fibronectin type III domains (FnIII1 to FnIII4) arranged in two pairs separated by a region na-med the connecting segment, downstream of the FnIII4 extends a 85-residue long C-terminal tail. It is proposed that prior to HD assembly the cytoplasmic domain of β4 adopts a closed conformation stabilized by an intramolecular interaction between the connecting segment and the tail; binding to plectin would unleash β4 and favour the association of β4 with other components of the HD [6]. Here we present the crystal structures the FnIII3 and FnIII4 domains, which have been refined to 1.6 and 1.8 Å of resolution, respectively. Analysis of the FnIII3-FnIII4 region by SAXS reveals a heart-shaped compact structure likely to correspond to the autoinhibited state of β4 in which the FnIII3-FnIII4 pair is bent to bring the connec-ting segment and the tail in close proximity. Finally, we have analysed the contribution of the connecting segment, the FnIII3 and FnIII4 to the interaction with BPAG1e.

[1] Tsuruta, D., Kobayashi, H., Imanishi, H., Sugawara, K., Ishii, M., and Jones, J.C. (2008). Current medi-cinal chemistry 15, 1968-1975.[2] Giancotti, F.G. (2007). Trends in pharmacological sciences 28, 506-511.[3] Wilhelmsen, K., Litjens, S.H., Kuikman, I., Tshimbalanga, N., Janssen, H., van den Bout, I., Raymond, K., and Sonnenberg, A. (2005). J Cell Biol 171, 799-810.[4] Koster, J., van Wilpe, S., Kuikman, I., Litjens, S.H., and Sonnenberg, A. (2004). Mol Biol Cell 15, 1211-1223.[5] de Pereda, J.M., Ortega, E., Alonso-Garcia, N., Gomez-Hernandez, M., and Sonnenberg, A. (2009). Cell Adh Migr 3, 361-364.[6] Koster, J., Borradori, L., and Sonnenberg, A. (2004). Handb Exp Pharmacol, 243-280.

contactauthor:noeliaalonsoInstituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones CientíficasUniversidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain..

113


poster 8

optical monitoring of single synaptic vesicle release from hippocampal neurons in cultureMª Ángeles Montes Fernández, Imane Jemal and Guillermo Álvarez de Toledo

Department of Physiology and Biophysics. School of Medicine. University of Seville. Avda. Sánchez Pizjuán4. Seville. Spain.

Neuronal communication resides in the information unit transfer conveyed by synaptic vesi-cles (SV) at synapses. A typical CNS synapse contains about 30–100 of these functional SV, but only a few fused with the presynaptic membrane upon arrival of an action potential. To determine the probability of vesicle fusion is thus critical to better understand the information transfer function of a synapse and to further study the mechanisms under its control. Here, we describe the direct optical detection of single quantal events in individual presynaptic buttons of culture hippocampal neurons transfected with a pH-sensitive fluorescence protein (superecliptic GFP) tagged to synaptophysin (SypHy). This probe serves as a functional tool of SV fusion since it is normally quenched in the acidic pH of the SV interior, but lights up upon exocytosis and exposure of the vesicle interior to the physiological pH of the synaptic cleft. To study the release probability of hippocampal neurons in culture, we have estimated the number of docked vesicles per active zone (5-10 vesicles). These docked vesicles corres-pond to the readily releasable pool. The arrival of an action potential at a synapse triggers neurotransmitter release with a release probability, p which define synaptic efficacy. With this approach we can determine the release probability of single synaptic boutons in hippocampal neurons and its variability within boutons from the same neuron. We have confirmed that re-lease probabilities for a single central synapse vary considerably from neuron to neuron and we find that most synapses have very low release probabilities, ranging from 0.19 to 0.30. We have further explored the quantal size distribution from single AP using low and high extracellular Ca2+concentration (1mM and 10mM) and we show that the release probability is increased at higher external Ca2+.

contactauthor:guillermoálVarezdeToledoDepartment of Physiology and Biophysics. School of Medicine. University of Seville. Avda. Sánchez Pizjuán4. Seville. Spain

114

poster 9

Functional organization of synaptic vesicles in hippocampal neurons monitored by fluorescent probesImane Jemal, María-Angeles Montes and Guillermo Alvarez de Toledo

Department of Physiology & Biophysics, School of Medicine. University of Seville. 41009 Seville. Spain

Synaptic vesicles (SV) are the key elements in neuronal communication since each vesicle release neurotransmitters in a quantal manner. Therefore to quantify the number of vesicles within a terminal and to determine their functional status is critical to further understand neuronal communication. Classically, SV reside in three different pools, reserve, recycling and readily releasable, in which the degree of recycling, positioning in the terminal and the inter-connectivity between them it has been a matter of debate for many years. Here we combine two optical probes, Sinaptophysin tagged to pHluorin `SypHy´ and the Styryl dye `FM4-64´, to image the life cycle of SV in hippocampal neurons in culture and electrically stimula-ted by an adjacent pipette. We have used Bafilomycin A1 to examine the partitioning of these probes into the different pools of SVs.

We show that a terminal contains on average about 92 vesicles spread between the recycling (53 vesicles, 54%) and the resting pool (40 vesicles, 46%).The fast releasable fraction of the recycling pool `readily releasable pool´ constitute a 7.4% of the total pool with an average of 9 vesicles per terminal and determine a release probability of 14% for a single vesicle fusion. These parameters vary from neuron to neuron. We have tried to identify the source of this variability. One critical point is the large variability of the total contents of vesicles (46 to 262). Another important aspect is the heterogeneous distribution of vesicles among different boutons. A fraction of the functional pool determines a synaptic efficacy and it doesn´t seem to be related with the spatial location of the terminal. An application of high external calcium does not vary the fraction of the pool but it reduces the range and makes synaptic boutons more homogenous. Finally, we see that the recycling pool is depleted exponentially sugges-ting a dynamic equilibrium between pools, allowing for compensation.

This assay can be used to further study the effects of genetic and chemical modulation of the SV cycle. The quantitative parameters determine here with live imaging could be used to elucidate the dynamic reorganization of synaptic vesicles pools and how this phenomenon relates to potentiating or depressing a synaptic terminal.

contactauthor:guillermoálVarezdeToledoDepartment of Physiology and Biophysics. School of Medicine. University of Seville. Avda. Sánchez Pizjuán4. Seville. Spain

115


poster 10

establishment of Drosophila melanogaster as a model system to study Multiple acyl Coa Dehydrogenation Deficiency (MaDD)Ema Alves1,2, Barbara J. Henriques2, Rui Gonçalo Martinho1, Cláudio Moreira Gomes2 1Instituto Gulbenkian de Ciência, Oeiras, Portugal2Instituto de Tecnologia Química e Biológica, Oeiras, Portugal

Multiple Acyl CoA dehydrogenation deficiency (MADD) is a disorder which implies mutations in one of the two mitochondrial genes that code for electron transfer flavoprotein (ETF) or electron transfer flavoprotein ubiquinone oxidoreductase (ETF:QO). These proteins are invol-ved in the pathway responsible for the metabolism of fatty acids, and, in such way, mutations could therefore lead to impairment and metabolic dysfunction with coupled mitochondrial failure. MADD can be grouped in three subtypes which refer to the neonatal lethal form with (type I), or without (type II) congenital anomalies and the milder form (type III) which is usually manifested latter in development and traduced in symptoms such as hypoglycaemia and liver failure.

We are currently working with three alleles of ETF:QO from Drosophila melanogaster. This protein is connected to the mitochondrial inner membrane where it is responsible for the transference of electrons to the ubiquinone pool of the electron transport chain (ETC). Bioche-mically, all the alleles under study, present mutations in the FAD domain. This fact could imply conformational changes as well as deficient electron transport which lead to the impairment of electron flux, thus interfering with the oxidation-reduction reactions that occur at both the β-oxidation pathway and ETC. Several activity assays, where citrate synthase activity was used as the reference control for the quality of mitochondrial preparation, showed remarka-ble differences in some of the measured parameters. The most prominent differences are observed for one of the proteins immediately upstream of ETF, the medium chain acyl CoA dehydrogenase (MCAD), for all the mutants and for NADH dehydrogenase , especially for the B42-1 mutant. In agreement with the expected destabilization of the FAD co-factor, essentially no activity of ETF:QO is found for neither of the mutants.

Analysis of the fatty acid metabolites by MS assays (generally used to screen newborns for metabolic disorders) revealed some alterations which are typical of fatty acid metabolic disor-ders thus giving emphasis to the results already obtained.

After cloning and expressing Drosophila ETF, we are currently expressing its partner ETF:QO. These steps are essential to carry out a comparative study with the human homologue pro-teins.

In conclusion, our main goal is to characterize these Drosophila ETF:QO mutants, compare them with the Human variants and finally, to establish Drosophila melanogaster as a valid model organism to study MADD.

contactauthor:Ema ALVESInstituto Gulbenkian de Ciência, Oeiras, Portugal

116

poster 11

a 2DCoS study of the effect of radiation on tgase activityN. ANDRAKA, I.DELAARADA1, J.GONZALEZ VELASCO2, P.BILBAO2,3 and J.L.R.ARRONDO11Unidad De Biofísica (Csic-Upv) And Departamento De Bioquímica, Universidad del País Vasco, Bilbao, Spain.2Depto. Cirugia, Radiología y Medicina Física , Universidad del País Vasco, Bilbao, Spain.3Servicio de Oncología Radioterapeútica, Hospital de Cruces-Osakidetza, Bilbao

Transglutaminases (TGase) are ubiquous enzymes activated only after major disruptions in physiological or homeostatic processes. However, their activity is strongly regulated since overexpression of the enzyme does not change cell survival. In mammalian cells 6 isozymes have been isolated even if the genome there are 8 different isozymes. The most characteristic is Factor XIII, involved in blood clotting activation. TGase has been isolated from keratino-cytes in soluble form or associated to membranes. The enzyme can be activated by physical agents such as ultraviolet or ionizing irradiation or by chemical agents such as the chemothe-rapeutic drugs. The changes produced in TGase activity under radiation have been attributed to metabolomic effects associated with variations in cell calcium levels. We have studied the changes in the presence of excess calcium and they are still present, pointing to a structural effect. In order to see the changes we have used TGase2 from hepatic cells being irradiated at different doses and measuring the amide I infrared spectrum at different times. As expected, since the cells irradiated are viable, the changes observed analyzing the decomposition are minimal, so we have used 2DCOS to analyze and visualize the changes. This approach shows that the changes observed in the amide I are consistent and allow us to propose that thee changes in activity produced in TGase upon radiation are due to structural changes rather to metabolomic alterations.

contactauthor:n.andraKaUnidad De Biofísica (Csic-Upv) And Departamento de Bioquímica Universidad del País Vasco, Bilbao, Spain.

117


poster 12

Fusion peptide effects on epitope recognition at Membrane Surfaces by the Broadly neutralizing anti-hiV-1 2F5 Monoclonal antibody Nerea Huarte1, Aitziber Araujo1, Rocío Arranz2, José M. Valpuesta2 and José L. Nieva1

1Biophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular Biology Department, University of the Basque Country, PO Box 644, 48080 Bilbao, Spain. 2Department of Macromolecular Structures, National Center for Biotechnology (CSIC), Canto-blanco, 28049 Madrid, Spain.

The HIV-1 glycoprotein fusogenic subunit gp41 is the target for 2F5, a broadly neutralizing monoclonal antibody (MAb2F5) isolated from asymptomatic infected individuals. The 2F5 epitope locates close to the membrane interface within the membrane proximal external re-gion (MPER) that connects the HIV-1 envelope gp41 ectodomain with the transmembrane anchor. Here evidence is presented indicating that the conserved amino-terminal fusion pep-tide (FP) increases the affinity of this antibody for its membrane-inserted epitope. Structural characterization by circular dichroism together with membrane-disrupting activity measure-ments suggests the formation of FP-MPER complexes at the surface of lipid bilayer vesicles. MAb2F5 associated more efficiently with lipid vesicles containing FP and MPER peptides as compared to those containing only MPER, or MPER in combination with FPctl, a scrambled version of the FP. Moreover, the N-terminal FP sequence had almost no effect on membrane-inserted epitope binding by MAb4E10, a neutralizing antibody that has been shown to extract its C-terminal MPER epitope from the membrane interface. In combination with recently re-ported crystallographic data (Julien et al. (2008) J. Mol. Biol., 384, 377-392), these results support a “catch-and-hold” mechanism for the process of MAb2F5-epitope binding at mem-brane surfaces.

contactauthor:aitziberaraujoBiophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular Biology Department, University of the Basque Country, PO Box 644, 48080 Bilbao, Spain.

118

poster 13

notable residues for the correct location of flavins in the n-terminal domain of FaD syntethase from C.ammoniagenes Sonia Susana Arilla Luna1,2, Adrián Velazquez-Campoy1,2 and Milagros Medina1,2

1Department for Biochemistry and Molecular and Cellular Biology, Faculty of Science, Univer-sity of Zaragoza 2BiFi: Institute for Biocomputation and Physics of Complex Systems, Zaragoza

FMN and FAD play critical roles as flavoprotein cofactors. Riboflavin (RF), their precursor, can be de novo synthesized or obtained from the environment in prokaryotes, yeast and plants, whereas only the second source applies for mammals. RF has to be transformed, first into FMN and then, converted into FAD, by the sequential action of two activities, an ATP:riboflavinkinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT) [1]. In eukaryotes the RFK and FMNAT activities reside in different proteins, while in most prokaryotes both activities are encoded by the same gene that produces a single protein. This protein, FAD syn-thetase (FADS), presents a C-terminal module showing high homology with RFKs. N-terminal module does not show similarity with eukaryotic FMNATs and rather belongs to the nucleo-tidylyl transferase superfamily [2]. A flavin site has been postulated at the FMNAT module [2][3]. Several mutants have been produced at this site in CaFADS. The role of each one of the mutated residues in the FMNAT activity has been established both by differential spectros-copy and isothermal titration calorimetry. The correct location of the flavin upon binding to FADS appears essential for the formation of a catalytically competent interaction.

[1] Efimov.I, Kuusk.V, Zhang.X & McIntire.WS (1998). Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli. Biochemistry, 37, 9716-9723.[2] Frago.S, Martínez-Júlvez.M, Serrano.A & Medina.M (2008). Structural analysis of FAD synthetase from Corynebacterium ammoniagenes. BMC Microbiology 2008, 8:160.[3] Frago.S, Velázquez-Campoy.A & Medina.M (2009). The puzzle of ligands binding to Corynebacterium ammoniagenes FAD synthetase. J.Biol.Chem 13;284(11):6610-9

contactauthor:soniasusanaarIllalunaDepartment for Biochemistry and Molecular and Cellular Biology Faculty of Science, University of Zaragoza.

119


poster 14

Lipid bilayer polarity features and thermal behavior in binary mixtures of popC, egg-sphingomyelin and cholesterolDalila Arrais1, Jorge Martins1,2

1IBB (Institute for Biotechnology and Bioengineering) - CBME (Center for Molecular and Structural Biomedicine)2DCBB - FCT, Universidade do Algarve, Campus de Gambelas, P-8005-139 Faro, Portugal

Cholesterol is one of the most biologically relevant lipid to be found namelly in plasmatic membranes. The addition of this molecule to pure phospholipid bilayers, besides the varia-tions in chemical composition, induces some important changes in the characteristic fluidity and polarity gradients [1]. Those changes lipid bilayers can also be exerted by other thermo-dynamic parameters (e.g. temperature, pressure) [2]. As already known, this will lead mostly to significant alterations in bilayer thickness, molecular packing (with a decrease on the con-formational freedom of the phospholipid acyl chains and the reduction of the lipid lateral diffusion) and water permeability.

We have studied the polarity properties and thermal behavior of pure phospholipid bilayers of pure POPC and egg-sphingomyelin (that are also two major constituents of the plasma-tic membranes) and of some specific compositions in POPC/cholesterol, egg-sphingomyelin/cholesterol and POPC/egg-sphingomyelin mixtures, using the Py empirical polarity scale [2]. We observed that with increasing cholesterol molar proportions in lipid bilayers, we obtain significantly different behaviors depending on the chemical composition: for POPC, there is a decrease of the polarity values which is similar to homogeneous polar solvents; for egg-sphin-gomyelin, there is an increase in those values, leading to results that show no relation with the classical behavior for homogenous solvents. For the POPC/egg-sphingomyelin mixtures, the results depend on which phospholipid is in excess.

[1] D. Marsh (2001) Proc. Natl. Acad. Sci. USA 98:7777-7782.[2] D. Arrais, J. Martins (2007) Biochim. Biophys. Acta – Biomembranes 1768:2914-2922.

Acknowledgments:

Dalila Arrais is recipient of a Ph.D. grant (SFRH/BD/41607/2007), from Fundação para a Ciência e a Tecnologia, Portugal.

contactauthor:dalilaarraIsIBB (Institute for Biotechnology and Bioengineering) - CBME (Center for Molecular and Structural Biomedicine)

120

poster 15

Molecular dynamics study of h2 diffusion in a [niFeSe]-hydrogenaseCarla Baltazar, Cláudio Soares1 1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal

Hydrogenases catalyze the reversible reaction H2 ⇆ H+ + e- [1]. They are believed to represent a solution for large scale oxidation and production of H2, which is regarded as a future alternative fuel. Nonetheless, some issues, like O2 inhibition, must be overcome for hydrogenases to be applied in technological systems.

Hydrogenases can be classified into [FeFe] or [NiFe] according to the metal content of the active site, and amongst them, some have particularly interesting properties. For instance, the [NiFeSe]-hydrogenases, a subgroup of [NiFe], are more tolerant toward O2 inhibition and present higher activities in H2 production [2]. They are the focus of the present work.

[NiFe]-hydrogenases, are globular proteins with a deeply buried active site, and H2 access and egress to or from this active site is made through hydrophobic channels identified in crystallogra-phic structures. Several studies indicate that altering the residues lining the channels can modulate the catalytic properties of the enzyme [3]. As the diffusion of H2 through the channels can not be followed directly by experimental means, molecular dynamics (MD) simulations are a good method to complement the knowledge on the field. In a previous study of H2 diffusion in the [NiFe]-hydro-genase from Desulfovibrio gigas, using explicit solvent MD, we were able to identify these channels and constricting residues [4].

In the work reported here, we aim at analyzing H2 permeation in [NiFeSe]-hydrogenases and com-pare the results with the ones previously obtained for [NiFe]-hydrogenases. We want to see if differences in protein architecture affect the H2 permeation process in a way that could relate to the observed differences in catalytical activity. In order to do that we simulated the diffusion of H2 in the structure of [NiFeSe]-hydrogenase from Desulfomicrobium baculatum [5] using MD methods. The hydrogenase was placed in a dodecahedric simulation box filled with SPC water, and 100 H2 mol were added to the solution, outside of the protein. In order to obtain reliable statistics, ten replicates of the system were simulated during 30ns, using GROMACS and the GROMOS 43A1 force field. Results show that the H2 molecules enter the protein through hydrophobic channels, in a similar way to what was previously observed with standard [NiFe]-hydrogenases [4]. However, small, but significant differences are observed in the substrate access behavior. These differences are analyzed and used to rationalize the different catalytic behavior between these two subgroups of hydrogenases.

[1] Vignais, P., Billoud, B., Occurrence (2007) Chem. Rev. 107, 4206-4272[2] Valente, F. M. A., Oliveira, A. S. F., Gnadt, N., Pacheco, I., Coelho, A. V., Xavier, A. V., Teixeira, M., Soares, C. M., Pereira, I. A. C. (2005) J. Biol. Inorg. Chem. 10, 667-682[3] Fontecilla-Camps, J.C., Volbeda, A., Cavazza, C., Nicolet, Y. (2007) Chem. Rev. 107, 4273-303[4] Teixeira, V. H., Baptista, M. A., Soares, C. M. (2006) Biophysical Journal 91, 2035[5] Garcin, E., Vernede, X., Hatchikian, E. C., Volbeda, A., Frey, M., Fontecilla-Camps, J.C. (1999) Structure 7, 557-566

contactauthor:carlaBalTazarInstituto de Tecnologia Química e Biológica Universidade Nova de Lisboa, Oeiras, Portugal

121


poster 16

Conformational Stability of the propeptide nh2-terminal of the precursor of pulmonary Surfactant protein B (Sp-B) to ph acidification and Chaotropes.Ángeles Bañares-Hidalgo, G. López, J. Pérez-Gil and P. Estrada

Department of Biochemistry and Molecular Biology, Faculty of Biology, Universidad Complu-tense de Madrid, José Antonio Novais 2, 28040 Madrid, Spain.

Pulmonary surfactant, a complex mixture of lipids and proteins, is necessary to prevent al-veolar collapse during expiration. Surfactant protein B (SP-B) is the most important protein to sustain surfactant function and is produced from the proteolytic processing of a precursor along the secretory pathway of surfactant in pneumocytes. The NH2-terminal flanking pro-peptide (SP-BN) is thought to act as chaperone of mature SP-B [1].

Previously, we have produced a recombinant form of SP-BN as a fusion protein with MBP in Escherichia coli, processed the fusion protein by proteolytic cleavage with Factor Xa and pu-rified SP-BN through ion-exchange chromatography. After a first molecular characterization of the propeptide [2] and having analyzed the conditions that promoted or hindered its self-aggregation [3], we have studied the chemical stability of the structure of the propeptide. To achieve this goal, we have analyzed the changes that chaotropes and pH acidification promote on the secondary and tertiary structure of SP-BN by means of circular dichroism and intrinsic fluorescence spectroscopy.

The secondary structure of the propeptide changed moderately upon shifting of pH from 7 to 4.1 and was much recovered after regaining neutrality. The unfolding transition of the propeptide in the presence of GdmCl or urea indicates that its chemical denaturation is not a two-state process and that an intermediate state is formed. Regarding the CD spectra, the midpoint transition determined from GdmCl and urea unfolding was 4.6 ± 0.2 M and 6.1 ± 1.9 M, respectively, and the apparent change in free energy obtained by LEM, 9 kJ•mol-1 and 11.6 kJ•mol-1, respectively. The relative low values of Gibbs energy point to the propeptide as being marginally stable. From the fluorescence spectra in GdmCl, the existence of an inter-mediate state was deduced, with a calculated midpoint for the intermediate-unfolded transi-tion at 4.3 ± 0.3 M and a change of free energy at zero GdmCl of 9.6 kJ•mol-1. Intermediate states with molten globule-like structure under both chemical and acidic denaturation were detected by means of extrinsic fluorescence spectroscopy using the probe bis-ANS.

[1] Lin S, Phillips KS, Wilder MR, Weaver TE (1996). Biochim Biophys Acta 1312:177-185.[2] Palacios A, González B, Alonso S, Pérez-Gil J, Estrada P (2006). Enzyme Microbiol Technol 40:85-92.[3] Bañares-Hidalgo A, Bolaños-Gutierrez A, Gil F, Cabré EJ, Pérez-Gil J, Estrada P (2008). J Ind Microbiol Technol 35:1367-1376.

contactauthor:ángelesBañares-hIdalgoDepartment of Biochemistry and Molecular Biology Faculty of Biology, Universidad Complutense de Madrid José Antonio Novais 2, 28040 Madrid, Spain.

122

poster 17

picornavirus 2B viroporin integrates into the er membrane as an α-helical hairpin Manuel Bañó1, Luis Martínez-Gil1 , Silvia Sánchez-Martinez2, José Luis Nieva2, Ismael Minga-rro1 1Departament de Bioquímica i Biología Molecular, Universitat de València 2Unidad de Biofísica y Departamento de Bioquímica, Universidad del País Vasco-CSIC

Virus infections can result in a range of cellular injuries and commonly these involve host membranes. Viroporin 2B has been identified as the viral protein that is responsible for the alterations in host cell membrane permeability that take place in enterovirus infected cells. Here, we show by in vitro translation of different fusion proteins carrying appropriate re-porter glycosylation tags that viroporin 2B is a double-spanning integral membrane protein inserted into the ER membrane through the translocon with an N-/C-terminal cytoplasmic orientation. In addition, the in vitro translation of several truncated versions of the tagged protein suggested that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes as an a-helical hairpin structure that apparently depends on specific helix-helix interactions.

contactauthor:ManuelBañóDepartament de Bioquímica i Biología Molecular Universitat de València

123


poster 18

Models of coupled molecular motors.Sebastián Bouzat1,2 and Fernando Falo1 1Departamento de Física de la Materia condensada and BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain. 2Consejo Nacional de Investigaciones Científicas y Técnicas, Centro Atómico Bariloche, (8400) Bariloche, Argentina.

We study theoretically the effect of interactions between molecular motors on the dynamics of a cargo pulled by multiple motors. We consider a realistic Monte Carlo model for the movement of individual motors, that considers the details of their coupling with the cargo and their detachment and attachment rates. It is based on previous models found in the li-terature [1,2], with the additional ingredients of interaction between motors and allowance of back motion. We find that motors interactions lead to a huge change in the velocity-force relation for the cargo. In particular, they produce a considerable decreasing of the stall force. Moreover, we show that the number of motors that effectively pull the cargo depends on the characteristics of the coupling spring that link them to the cargo. We study the load--velocity curves, the run lengths, and the distribution of forces on the different motors, comparing results with those from models with non detaching and non interacting motors.

[1] Kunwar, A.,Vershinin, M., Xu, J., and Gross, S.P. (2008) Current Biology 18, 1173. [2] Kunwar, A. and Mogilner, A. (2010) Phys. Biol. 7, 016012.

contactauthor:sebastiánBouzaTDepartamento de Física de la Materia condensada and BIFI, Universidad de Zaragoza 50009 Zaragoza, Spain.

124

poster 19

impact of subdiffusion on the formation and properties of morphogen gradients. a reaction-diffusion approach.Santos Bravo Yuste1, Enrique Abad1, and Katja Lindenberg2

1Dpt. Física. Univ. Extremadura. E-06071, Badajoz, Spain 2Dpt. of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0340, USA

Morphogen gradient is a key mechanism in developmental biology by which signaling mo-lecules (the morphogens) emitted from one part of an embryo determine the location and fate of the surrounding cells in a concentration-dependent manner [1]. A standard way of explaining its formation is a reaction-diffusion model where a source of normal diffusive mor-phogens is coupled with a degradation mechanism that removes them from the system [1,2]. However, it is well-known that biological media are crowded environments where diffusion is often anomalous [3]. In particular, diffusion is often subdiffusive. In this context, we address the question of how the subdiffusive character of the morphogens affects the formation and properties of the morphogen gradient. In contrast with some previous models, we show by means of a reaction-subdiffusion model, derived directly from a CTRW description of the mor-phogens, that subdiffusive morphogens are compatible with the development of a stationary profile even within the basic and usual scenario where the morphogen degradation is linear. Interestingly, we show that, at odds with normal diffusion, there is no stationary profile if the degradation rate is zero or goes to zero exponentially outside of a finite region that encloses the source of particles. We also discuss how the subdiffusive character of the morphogens affects the robustness of their profile against changes in the degradation rate and secreted flux.

[1] Ibáñez, M., and Izpisúa, J.C. (2008) Mol. Syst. Biol. 4:176.[2] Yu, S.R., Burkhardt, M., Nowak, M., Ries, J., Petrasek, Z., Scholpp, S., Schwille, P., and Brand, M. (2009) Nature 461, 533-536.[3] Goldin, I., and Cox, E. C. (2006), Phys. Rev. Lett. 96, 098102.[4] Dix, J. A., and Verkman, A. S. (2008) Annu. Rev. Biophys. 37, 247-263.

contactauthor:santosBraVoyusTeDpt. Física. Univ. Extremadura. E-06071, Badajoz, Spain

125


poster 20

Modeling the folding of gpW and alpha-spectrin Sh3 domain: accurate information from a simple model.Pierpaolo Bruscolini1, Athi Narayanan Naganathan2

1Instituto de Biocomputación y Física de de Sistemas Complejos (BIFI) & Departamento de Física Teórica, Universidad de Zaragoza, Zaragoza, España2Institute for Research in Biomedicine, Barcelona, España

We extend the WSME model, to include the phenomenological information coming from Freire’s results on the different contributions to protein specific heat. We adjust the four pa-rameters of the resulting model by fitting the raw calorimetric data for gpW and SH3 domain, without baseline subtraction, to avoid the known problems relative to baseline identification for weakly cooperative proteins.

We use the resulting model to describe both the thermodynamics (through exact solution) and the kinetics (with MC simulations) of the proteins, with a special attention to gpW, and we are able to reproduce several of the experimental features, both for thermodynamics and kinetics. Our results show that, despite the similar size and mid-folding temperature, the two proteins show very different behaviors: while the SH3 domain appears as a clear two-state protein, gpW shows a free-energy landscape with small barriers (around 5 kJ/mol), and a kinetics which presents a slow phase, corresponding to folding, and a fast phase, of re-arran-gements within the initial basin.

contactauthor:pierpaoloBruscolInIInstituto de Biocomputación y Física de de Sistemas Complejos (BIFI) & Departamento de Física Teórica, Universidad de Zaragoza, Zaragoza, España

126

poster 21

puzzling out the Mechanical Stability of the Dorsal-Ventral organizer in the Wing imaginal Disc of DrosophilaOriol Canela Xandri1, Francesc Sagués2, Jaume Casademunt3, and Javier Buceta1

1Computer Simulation and Modelling (Co.S.Mo.) Lab, Parc Cientific de Barcelona, C/ Baldiri Reixac 10, Barcelona 08028 Spain 2Departament de Química-Física, Universitat de Barcelona, C/ Martí Franqués 1, Barcelona 08028, Spain3Departament de Estructura i Constituents de la Materia, Universitat de Barcelona, C/ Martí Franqués 1, Barcelona 08028, Spain

Dorsal ventral boundary formation is crucial in the establishment of the body plan during wing formation in Drosophila. This process is different to other processes of boundary for-mation as the AP boundary since a specialized boundary cell population (the fence) becomes specified in this case. By using a dynamical vertex model and a realistic description of the cell cycle and the division processes that includes stochastic variability, herein we address the problem of how the mechanical stability of the DV boundary is achieved in order to restrict the cellular lineages of the D and V compartments.

leukocytes triggers molecular and biophysical consequences in Kv1.3/Kv1.5 hybrid channels by altering the subunit stoichiometry.

Supported by BFU2008-00431 and CSD2008-00005 from MICINN (Spain)

contactauthor:Javier BUCETA

127


poster 22

Molecular dynamics simulations of Dna melting in the presence and absence of some antitumour drugs covalently bonded in the minor grooveJuan A. Bueren-Calabuig,1 Carlos M. Galmarini,2 Carmen Cuevas2 and Federico Gago11Departamento de Farmacología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid.2PharmaMar, S.A.U. Pol. Ind. La Mina, 28770 Colmenar Viejo, Madrid.

In a standard DNA double helix two complementary polynucleotides interact with each other via Watson-Crick (WC) hydrogen bonds. The properties of a double-stranded (ds) DNA de-pend on the base composition and sequence of the phosphodiester chain and can be strongly perturbed by alterations in pH, temperature and solvent. Besides, in living cells, protein-me-diated physical disruption of dsDNA and local formation of single-stranded (ss) regions are necessary for normal DNA replication, transcription and repair. These vital processes can be affected by antitumour drugs including the well-known interstrand crosslinker mitomycin C and the monofunctional DNA-binders Yondelis® and Zalypsis®. Monoadducts involving these latter drugs have been shown to increase substantially the melting temperature of selected DNA oligonucleotides [1,2]. These three compounds covalently bind to the exocyclic N2 of a guanine in the DNA minor groove. To study their effects on the DNA melting process, a 15-mer dsDNA of sequence d(TAATAACGGATTATT)•d(AATAATCCGTAATTA) was simulated at 400 K and 0.1 M NaCl [3] using molecular dynamics (MD) and the AMBER force field [4] both in the absence and in the presence of bonded drugs. Yondelis® and Zalypsis® were covalently bonded to the underlined guanine in the first strand whereas the covalent adduct with mi-tomycin C linked together this guanine and the only guanine in the opposite strand. After 200 ns of MD simulation, all the original WC hydrogen bonds were lost in the free DNA whereas an extended WC-paired region embedding the central CGG triplet was still maintained in all drug-DNA complexes, which attests to the ability of these drugs to stabilize dsDNA and stall replication and transcription forks. Furthermore, some reannealing events were observed in the complexes with Yondelis® and Zalypsis®. Taken together our findings support the view that the adducts formed by these two drugs in a DNA molecule functionally behave as inters-trand crosslinks.

[1] Casado, J.A., Río P., Marco E., García-Hernández V., Domingo A., Pérez L., Tercero JC., Vaquero J.J., Albella B., Gago F., Bueren J.A. (2008) Mol Cancer Ther. May;7(5):1309-18.[2] Leal J.F., García-Hernández V., Moneo V., Domingo A., Bueren-Calabuig J.A., Negri A., Gago F., Guillén-Navarro M.J., Avilés P., Cuevas C., García-Fernández L.F., Galmarini C.M. (2009) Biochem Pharmacol. 78, 162-170.[3] Wong, KY., and Pettitt, M. (2008) Biophys. J. 95, 5618-5626.[4] http://ambermd.org

contactauthor:juana.Bueren-calaBuIgDepartamento de Farmacología, Universidad de Alcalá 28871 Alcalá de Henares, Madrid

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128

poster 23

aFM Study of the interaction of a synthetic peptide from de gB virus C/ hepatits g virus with the hiV fusion peptideÒscar Domènech, Josefina Prat, M. Antònia Busquets, M. Asunción Alsina, Victòria Girona

Physicalchemistry Department. IN2UB. Associated Unit to the CSIC: Peptides and proteins: Physicochemical studies. Faculty of Pharmacy. University of Barcelona. Avda Joan XXIII, s/n 08028 Barcelona.

AIDS is a disease that has become pandemic on late 20th century. During years, studies on HIV have developed enough knowledge about the disease to decrease mortality on patients. Usually patients with HIV acquire other infections that can cause death, but others infections, as Hepatitis G (HGV), has demonstrated a decrease of virulence of HIV [1, 2]. Authors suggest HGV inhibits the replication of HIV but mechanism is not well understood. It is suggested HGV has a potential inhibition on the fusion of HIV to cells.

To evaluate this possible inhibition our group investigated morphological changes of two peptide fragments: GP41 from HGV and P59 from HIV peptide fusion. Studies were carried out with the Atomic Force Microscope to visualize the interaction of peptides with themsel-ves and when they were mixed. AFM images of GP41 and images of P59 showed spherical structures on mica surface suggesting aggregation of peptide when they were deposited on a flat surface. On the other hand, when both peptides were incubated together at a ratio of 1:5 (GP41:P59, mol/mol) annular structures of 200 nm of diameter appeared on mica surface. These structures were aligned forming chains were each link could be the association of one GP41 peptide surrounded by five P59 peptides.

These annular structures suggest morphological changes on the organization of peptides due to the exposure of regions towards media that were not exposed before. The new organization should be due to the protection of these regions towards a more stable structure in the media.

[1] Tillmann HL, Heiken H, Knapik-Botor A, et al. (2001) N. Engl. J. Med.; 345, 715–24.[2] Williams CF, Klinzman D, Yamashita TE, et al. (2004) N. Engl. J. Med.; 350, 981–90.

Acknowledgements. This work was supported by Grants CTQ2009-13969-C02-01/02 from the Ministerio de Ciencia e Innovación and by 2009SGR560 from the Generalitat de Catalun-ya (Spain).

contactauthor:antòniaBusqueTsPhysicalchemistry Department. IN2UB. Associated Unit to the CSIC: Peptides and proteins: Physico-chemical studies. Faculty of Pharmacy. University of Barcelona. Avda Joan XXIII, s/n 08028 Barcelona.

129


poster 24

regulation of endocytotic fission pores by calcium and dynamin José María Cabeza1, Jorge Acosta1 & Eva Alés1

1Departamento de Fisiología Médica y Biofísica. Facultad de Medicina. Universidad de Sevilla.

Although endocytosis involves the fission pore, a transient structure that produces the scis-sion between vesicle and plasma membranes, the dimensions and dynamics of fission pores remain unclear. Here we report that membrane scission occurs through sequential stages that involve changes, first in pore diameter and then in pore length. The kinetics of these changes were calcium-dependent. Before fission, the pore diameter consistently decreased to a value near 4 nm, whereas the pore length ranged between 20- 300 nm and was influen-ced by the intracellular calcium concentration. Dynamin, a mechanochemical GTPase, plays an important role in accelerating the fission event, preferentially in endocytotic vesicles of regular size, by increasing the rate of narrowing and lengthening of fission pores, but hardly affected larger and longer-lived endocytotic events. These results suggest fission pores are dynamic structures that form thin and long membrane necks regulated by intracellular cal-cium. Between calcium mediators, dynamin functions as a catalyst to increase the speed of single vesicle endocytosis.

contactauthor:joséMaríacaBezaDepartamento de Fisiología Médica y Biofísica Facultad de Medicina. Universidad de Sevilla.

130

poster 25

Membrane topology and protein-protein interactions of pulmonary surfactant protein Sp-B studied by fluorescence spectroscopy Elisa J. Cabré1, Luís M. S. Loura2,3, Alexander Fedorov4, Jesus Perez-Gil1, Manuel Prieto4

1Dept. Bioquímica, Fac. Biología, Universidad Complutense, 28040 Madrid, Spain. 2Faculdade de Farmácia, Universidade de Coimbra, 3000-548 Coimbra, Portugal.3Centro de Química de Coimbra, 3004-535 Coimbra, Portugal.4Centro de Química-Física Molecular , Instituto Superior Técnico, and IN-Instituto de Nano-ciências e Nanotecnologias, 1049-0001 Lisboa, Portugal.

Pulmonary surfactant is a lipid-protein complex synthesized and secreted by the alveolar epithelium of lungs, which main function is to reduce surface tension at the air-liquid interface, stabilizing the res-piratory surface against collapse at the end of expiration [1, 2]. Pulmonary surfactant is composed of roughly 90% lipids and 8-10% of specific surfactant-associated proteins, termed in chronological order of discovery SP-A, SP-B, SP-C and SP-D [3]. SP-B is involved in the transfer of phospholipid molecules from specific lipid/protein assemblies produced by pneumocytes into the surface active interfacial film. The lack of SP-B is lethal, being its absence associated with an irreversible respiratory failure at birth. Presence of hydrophobic proteins SP-B and SP-C is strictly required to facilitate an appropriate stability of the surface-active films along the breathing cycles [4]. Most therapeutic surfactant preparations cu-rrently in use in the treatment of respiratory pathologies are obtained from animal sources and contain variable amounts of SP-B and SP-C. The molecular mechanisms by which surfactant proteins cooperate in the assembly, transport, and reorganization of surfactant lipids at the respiratory surface are still not well understood.

To obtain further details on the orientation of protein SP-B in phospholipid membranes, we have analy-zed the fit of FRET data obtained by using the single tryptophan in SP-B as a donor and different NBD-labeled lipids as acceptors, to two different potential models of how SP-B could be orientated with respect to phospholipid membrane surfaces. Our results are consistent with topology models in which the pro-tein has a shallow orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction.

Furthermore, we have studied the effect of the presence of the other hydrophobic protein in surfactant, SP-C, on the fluorescence of SP-B in membranes. Self-quenching analysis of intrinsic and extrinsic (Alexa-labeled) fluorescence of SP-B concludes the existence of SP-B homo-oligomerization and the participation of the protein in high order oligomers. The dependence of quenching of SP-B fluorescence on the concen-tration of SP-C in the membrane reveals SP-B/SP-C interaction.

[1] Pérez-Gil, J.Biochim Biophys Acta, 2008. 1778: 1676-95.[2] Zuo, Y.Y., et al. Biochim Biophys Acta, 2008. 1778: 1947-77. [3] Johansson, J. and T. Curstedt. Eur J Biochem, 1997. 244: 675-93.[4] Serrano, A. G. and J. Perez-Gil. Chem. Phys. Lipids, 2006. 141: 105-118.

contactauthor:elisaj.caBréDept. Bioquímica, Fac. BiologíaUniversidad Complutense, 28040 Madrid, Spain.

131


poster 26

pharmacological chaperones for the aromatic amino acid hydroxylasesAna C. Calvo1, Tanja Scherer2, Angel L. Pey1, Ming Ying1, Ingeborg Winge1, Jeffrey McKinney1, Knut Teigen1, Jan Haavik1, Beat Thöny2 and Aurora Martinez11Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway2Department of Pediatrics, University of Zürich, Zürich, Switzerland

The aromatic amino acid hydroxylase (AAAH) enzyme family includes phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and the tryptophan hydroxylases (TPH-1 and 2). These enzymes are structurally and functionally related and in mammals they are mainly distributed in liver and kidney (PAH) and neuronal and neuroendocrine tissues (TH and TPH-1 and 2). The AAAHs share a number of ligands, such as the cofactor tetrahydrobiopterin (BH4) and inhibitors. In fact the AAAHs do not show strict substrate selectivity, and can utilize all three aromatic amino acids as substrates to some extent. It is thus important to investigate binding selectivity for compounds used in therapeutic interventions aiming the modulation of each AAAH, such as BH4, stabilizers (pharmacological chaperones) and inhibitors. Through high-throughput screening we recently discovered four compounds (I-IV) that functioned as pharmacological chaperones for PAH (Pey et al. (2008) J Clin Invest 118, 2858-2867). These compounds stabilized the functional tetrameric conformation of recombinant wild-type PAH and PKU mutants, and increased steady-state PAH protein levels in eukaryote cells and liver of treated mice. We now investigated the effect of these compounds on TH and TPH2 in vitro, comparative to PAH, by differential scanning fluorimetry. For the most effective compounds (II-IV) we also measured their in vivo effect. Only compound III (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) bound and stabilized all three purified enzymes (PAH, TH (isoform 1) and TPH2), and increased TH activity in treated mice. For the other compounds, different effects were measured for their action in vitro and in vivo for each AAAH. Our results will be discussed in the context of the potential of these compounds as pharmacological chaperones for the treatment of disorders associated with AAAH misfolding.

contactauthor:anac.calVoDepartment of Biomedicine University of Bergen, N-5009 Bergen, Norway

132

poster 27

global Downhill vs. Barrier-Limited Folding in Single MoleculesLuis Alberto Campos-Prieto1, Jianwei Liu3, Ravishankar Ramanathan1, Xiang Wang4, Douglas English5 & Victor Muñoz1,2

1Centro de Investigaciones Biológicas-CSIC,Ramiro de Maeztu 9,Madrid 28040,Spain.2Dept. of Chemistry & Biochemistry,Univ. of Maryland,College Park,MD 20742,USA.3Dept. of Chemistry,Stanford Univ.,333 Campus Drive,Stanford,CA 94305-5080,USA.4Dept. of Chemistry,Univ. of North Carolina,Chapel Hill,NC 27599-3290,USA.5Dept. of Chemistry,Wichita State Univ.,1845 Fairmount St.,Wichita, KS 67260,USA.

One-state downhill folding is characterized by a free energy surface with a single well, which in native conditions is placed at high values of the reaction coordinate corresponding to a defined 3D structure but shifts progressively to higher disorder upon increasing denaturational stress. One-state folding proteins could exploit their rich conformational behavior as molecular rheostats [1]. Downhill folding is thus an exciting problem for single molecule (SM) spectroscopy, which, in principle, could resolve the characteristic unimodal distribution of protein conformations shifting from order to disorder as a function of denaturant. However, one-state downhill folding should be very fast, with relaxation times of a few microseconds at room temperature, placing it well beyond the currently accessible timescales of free diffusion SM FRET experiments.

Here we approach this problem studying the chemical unfolding of the small αμ-helical protein BBL, a microsecond folding protein that has been characterized as a one-state downhill folder based on a battery of ensemble equilibrium and kinetic experiments [2,3]. Other groups, however, have claimed that BBL folds crossing a large folding barrier using qualitative data without further analy-sis from SM FRET, also raising issues about possible artifactual effects from tail truncation and/or partial histidine protonation [4,5]. To tackle the time-resolution limitation we have performed single molecule FRET experiments at 278 K, which slows BBL folding down to ~120 microseconds, and developed a new cocktail of triplet quenchers and oxygen-radical scavengers to obtain SM ave-rage fluxes in the 0.5-0.7 photons/ms range. Free diffusion experiments with a binning time of 50 µs produce unimodal FRET efficiency distributions with a maximum that shifts monotonically from high to low FRET values as the concentration of chemical denaturant increases. These experiments unravel the one-state downhill folding of BBL at the single molecule level opening exciting possi-bilities for SM experiments. Furthermore, the experiments demonstrate that one-state downhill folding is robust feature of the BBL protein and the importance of thorough quantitative analysis of the SM data.

[1] Garcia-Mira, M.M., Sadqi, M., Fischer, N., Sanchez-Ruiz, J.M. & Muñoz, V. (2002). Science 298(5601), 2191-2195.[2] Sadqi, M., Fushman, D. & Muñoz, V. (2006). Nature 442(7100), 317-321.[3] Li, P., Oliva, F.Y., Naganathan, A.N. & Muñoz, V. (2009). Proc. Natl. Acad. Sci. U.S.A. 106(1), 103-108.[4] Arbely, E., Rutherford, T.J., Sharpe, T.D., Ferguson, N. & Fersht, A.R. (2009). J. Mol. Biol. 387(4), 986-992.[5] Huang, F., Ying, L. & Fersht, A.R. (2009). Proc. Natl. Acad. Sci. U.S.A. 106(38), 16239-16244.

contactauthor:luisalbertocaMpos-prIeToCentro de Investigaciones Biológicas-CSIC Ramiro de Maeztu 9,Madrid 28040,Spain..

133


poster 28

active Zone organization and distribution of synaptic vesicles in the neuromuscular junction of Synaptophysin-phluorin transgenic miceRaquel Cano1, Rocío Ruiz1, Juan José Casañas1, William J. Betz2 and Lucía Tabares1 1Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, 41009, España.2Department of Physiology and Biophysics, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 (USA)

The precise location of synaptic vesicles in active zones (AZs), specialized regions where synaptic vesicles fusion with the plasma membrane takes place, is a highly regulated pro-cess which involves a large number of proteins such as Bassoon, Piccolo, RIM, etc. The use of pHluorins (a GFP particularly sensitive to pH changes) attached to the lumenal side of synaptic vesicle membrane proteins allows us to examine both the exo-endocytosis and the distribution of vesicles in the presynaptic terminal. In our laboratory we have designed a new line of transgenic mice (Syph) expressing a fusion protein consisting on synaptophysin and pHluorin, under the neuron specific promoter Thy 1.2 (“thymus cell antigen 1”). Syph is expressed in the transgenic mouse neuromuscular junction, and colocalizes with SV2 (ano-ther synaptic vesicle protein), as assessed by immunostaining, indicating that the chimeric protein is correctly localized in synaptic vesicles. We also studied the organization of the AZs with antibodies against Bassoon and Piccolo, two scaffolding proteins of AZs, and found that the mean number of AZs per terminal is 800, its density is 2.4 / μm2, and that the average minimum distance between neighbour AZs is 600 nm. We have also found that AZ proteins are juxtaposed with the postsynaptic folds, rich in cholinergic nicotinic receptors. These tools allow us to study in a terminal the distribution of vesicles, the organization of the AZs and the relationship between them, both at rest and following electrical activity.

contactauthor:raquelcanoDepartamento de Fisiología Médica y Biofísica, Facultad de Medicina Universidad de Sevilla, 41009, España..

134

poster 29

protein-structure-based dot plotOscar Conchillo-Sole1 Xavier Daura1,2

1Universitat Autònoma de Barcelona, Barcelona, Spain2Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain

Dot plots were first introduced as a bioinformatics technique in 1981 by Maizel and Lenk[1]. Their goal was to create a simple way to visualize similarities between two protein sequences. Dot plots are two-dimensional identity matrices, in this case applied to residue sequence. Thus, if two proteins are identical a diagonal crossing the matrix will be observed. If the se-quences contain repeats off-centre diagonals will be observed and if those repeats are inver-ted these diagonals will be inverted too. Blanks in the diagonal indicate regions with different sequence and displacements indicate insertions or deletions.

Proteins may not only be compared by their sequence, but structure is an additional indica-tor of similarity or relationship. When comparing structures what matters is not the type of amino acid present in a specific position in sequence space, but the relative position of this residue in 3D Cartesian space (or an equivalent space of internal coordinates). For example, if we define the position of each residue with respect to the closest ones in the sequence, those groups of residues having the same internal distances and angles in two different proteins can be said to share the same local structure. Global similarity is then evaluated from the succession of local similarities.

To apply a method based on local structure such as this, it is necessary to determine the grou-ping of residues that is going to be used for the comparison. Thus, for each possible group of four consecutive residues in the protein sequence, three distances and three angles are calcu-lated between the respective alpha-carbons and assigned to the second residue. As a result, every residue in the sequence (except the first and the last two) is characterized by a set of six internal coordinates, which may be used directly, in a dot-plot fashion, to compare proteins at the structure level and assess similarity according to a defined threshold.

This method is still under development, but has already been used to compare a number of different loops with very promising results, i.e. revealing similarities between them that otherwise would remain hidden.

References[1] J. V. Maizel, Jr. and R. P. Lenk. Enhanced graphic matrix analysis of nucleic acid and protein sequen-ces Proc. Natt Acad. Sci. USA 1981, 78(12): 7665-7669.

contactauthor:oscarconchIllo

135


poster 30

adenovirus polypeptide iVa2: looking for a new viral portal Menéndez-Conejero, R.1; Pérez-Berná, A.J.1; Ostapchuk, P.2; Hearing, P. 2; San Martín, C.1

1Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Darwin, 3; 28049 Madrid, Spain2Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11794

Abstract IVa2 is a key piece in adenovirus morphogenesis. This minor component binds spe-cifically to the viral DNA packaging domain [1, 2] and contains Walker A and B consensus motifs indicating that it may have ATPase activity; has been shown to bind ATP [3] and to be located at a single capsid vertex [4]. All these features suggest that IVa2 may be the adenovirus packaging motor, by analogy with tailed bacteriophages. Using far Western blot and cross-linking assays we have determined that recombinant IVa2 has the ability to homo-oligomerize. Electron microscopy indicates that the oligomeric form is a ring. The outer dia-meter of the ring is 12 nm, comparable to the diameter of the outermost domain of penton base. The inner diameter of the ring is approximately 4 nm, so dsDNA could be transported through this channel. Thus, adenovirus IVa2 shares a key structural arrangement with other packaging portals and DNA translocases.

[1] Zhang, W. and Imperiale, M.J. (2000) Interaction of the adenovirus IVa2 protein with viral packaging sequences. J Virol, 74, 2687-2693.[2] Zhang, W., Low, J.A., Christensen, J.B. and Imperiale, M.J. (2001) Role for the adenovirus IVa2 protein in packaging of viral DNA. J Virol, 75, 10446-10454.[3] Christensen, J.B., Byrd, S.A., Walker, A.K., Strahler, J.R., Andrews, P.C. and Imperiale, M.J. (2008) Presence of the adenovirus IVa2 protein at a single vertex of the mature virion. J Virol, 82, 9086-9093.[4] Ostapchuk, P. and Hearing, P. (2008) Adenovirus IVa2 protein binds ATP. J Virol, 82, 10290-10294.

contactauthor:Menéndez-conejero,r.

136

poster 31

Lipid-protein interaction: quantifying protein binding to lipid vesicles using single colour fluorescence correlation spectroscopy (FCS)Ana Coutinho1,2, Ana Melo1 and Manuel Prieto1

1Centro de Química Física Molecular, Complexo I, Instituto Superior Técnico, and Institute of Nanosciences and Nanotechnology, 1049-001 Lisboa, Portugal2Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de 1749-016 Lisboa, Portugal

Quantitative evaluation of peptide/protein binding to liposomes through the determination of its partition coefficient is an important step in any lipid-protein interaction study because it allows the calculation of peptide/protein interfacial coverage of the lipid vesicles, often the critical parameter controlling the protein membrane binding mode (peripheral vs partial insertion) and/or its oligomerization state. Fluorescence correlation spectroscopy (FCS) is a technique with single-molecule sensitivity that has been recognized as a reliable method to achieve this goal [1]. In this work, we used FCS binding data obtained for lysozyme labelled with Alexa Fluor 488 and extruded liposomes prepared with variable anionic lipid content to illustrate some potential pitfalls in data analysis that can lead to an overestimation of these coefficients. The observable species in a FCS measurement are the free fluorescently-labelled protein and the lipid vesicles with one or more bound conjugated proteins, characterized by very different diffusion coefficients. Considering that in a multicomponent system the mea-sured autocorrelation function is a weighted sum of the autocorrelation functions of each component, with amplitudes proportional to the square of the molecular brightness of the diffusing particles detected, we will show first the need to consider explicitly the mean occu-pancy number of the lipid vesicles by the conjugated protein through the use of a Poisson dis-tribution when the experimental autocorrelation curves are analyzed using a fitting function with fractional amplitudes. Secondly, we will discuss how the presence of a very small amount of free fluorescent dye (nonbinding component) in the system can affect the experimental binding curves and, consequently, the recovered protein partition coefficients.

Fundação para a Ciência e Tecnologia (FCT) is acknowledged for financial support (Project PTDC/QUI-BIQ/099947/2008 and Ph.D. grant SFRH/BD/61723/2009 to Ana Melo).

[1] Rusu, L., Gambhir, A., McLaughlin, S., and Rädler, J. (2004) Biophys. J. 87, 1044-1053.

contactauthor:anacouTInho

137


poster 32

Dynamic interconversion of oligomeric and fibrillar forms of alpha-synuclein Nunilo Cremades1,2, Allen Y. Chen1, Angel Orte3, Massimo Sandal1, Paul Dunne1, Francesco A. Aprile1, Richard Clarke1, Carlos W. Bertoncini1, Christopher M. Dobson1, and David Klener-man1.

1Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.2Institute for Biocomputation and Physics of Complex Systems. University of Zaragoza, Zara-goza, Spain3Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Granada, Spain

Revealing the key molecular processes by which proteins misfold first into soluble oligomers and then into amyloid fibrils is an essential first step to understanding neurodegenerative disorders such as Alzheimer’s and Parkinson´s disease, especially after all the accumulating evidence suggesting that soluble oligomers may be responsible for neuronal death. Achieving this objective, however, has proved challenging, particularly since the key species at the early stages of the reaction are present at low concentrations and can be heterogeneous both in size and conformation. We have used single-molecule intermolecular FRET to simultaneously obtain information on the structure and size of individual oligomers formed during the pro-cess of aggregation and disaggregation of alpha-synuclein, the protein whose deposition in the brain is associated with Parkinson’s disease. We find the initial rate limiting step from the monomeric protein to be a conformational change which leads to the formation of oligomers, and that a structural reorganisation within 10mer oligomers from unstructured to beta-sheet, fibrillar-like structure then required for further assemble into fibrils. In spite of the hetero-geneous distribution of oligomers in size, only two main structural populations of oligomers are populated during alpha-synuclein fibrillization. Both types of oligomers are also present under physiological concentrations, when only monomers were thought to be present. The various oligomeric species are in dynamic equilibrium with the fibrils, a situation that sug-gests that the fibrils act either to sequester or release potential toxic species depending on the conditions.

contactauthor:nunilocreMades

138

poster 33

atomic force microscopy of oligomeric arrangements of surfactant protein Sp-B on Langmuir-Blodgett films.Antonio Cruz, Bárbara Olmeda and Jesús Pérez-Gil

Depto. de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense de Madrid, SPAIN

Pulmonary surfactant protein SP-B is the only surfactant protein that has been demonstrated to be essential for the initiation and maintenance of breathing. The absence of the protein in mice with the SP-B gene knocked-out is lethal shortly after birth [1]. SP-B is synthesized by the type II cells of the alveolar epithelium and secreted to the extracellular medium at the air spaces of lungs together with surfactant lipids. Proposed functions for SP-B include to promo-te a very rapid adsorption of surfactant phospholipids into the pulmonary air-liquid interface and to maintain the surface-associated structures fully functional along the compression-ex-pansion respiratory cycling [2]. Mature SP-B is a 9 kDa (79 aminoacids) peptide belonging to the family of saposin-like proteins (SAPLIP), characterized by a high content of alpha-helical secondary structure and by the presence of six conserved cysteine residues forming three intramolecular disulfide bridges [2]. SAPLIP family is constituted by several proteins with diverse functions including: (i) membrane targeting, (ii) presentation of lipids as substrates for independent enzymes and (iii) membrane permeabilization by perturbation either as mo-nomer or by formation of oligomeric pores [3]. Unlike other SAPLIP proteins, SP-B is highly hydrophobic and permanently associated with lipids and forms homodimers stabilized by an additional intermolecular disulfide bridge.

Interfacial monolayers of pure SP-B were compressed at different surface pressures and then transferred onto mica supports to obtain Langmuir-Blodgett (LB) films of the protein. Atomic force microscopy (AFM) allowed detecting the organization of macromolecular aggregates of the protein in the LB films, which were compatible with defined oligomeric arrangements of the protein. The addition of small amounts of phospholipids to the protein prior to the interfacial monolayer preparation gives rise to the orientation of the protein particles at the air-liquid interface, making possible the observation of discrete units with regular sizes and shapes.

[1] Clark, J. C., Wert, S. E., Bachurski, C. J., Stahlman, M. T., Stripp, B. R., Weaver, T. E., and Whitsett, J. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7794-7798.[2] Serrano, A. G. and Perez-Gil, J. (2006) Chem. Phys. Lipids 141, 105-118.[3] Bruhn, H. (2005) Biochem. J. 389, 249-257.

contactauthor:antoniocruz

139


poster 34

aBtS interaction and electron transfer with Cota laccase: a molecular modelling approachJoão M. Damas, Cláudio M. Soares

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal

Laccases are enzymes that belong to the multicopper oxidase family. These enzymes couple the reduction of dioxygen to water in a trinuclear copper centre, the T2/T3 centre, with the oxidation of a substrate in a mononuclear copper centre, the T1 centre [1]. Their biotechnolo-gical importance is large and their industrial applications are diverse, mainly due their broad substrate specificity that can be augmented by the use of redox mediators [2].

Despite the fact that the biological substrate of laccase is not known yet, ABTS is widely used as a laccase substrate and was the first laccase redox mediator. Its oxidation kinetics and me-chanism has been studied through spectroscopic and electrochemical techniques [3,4], but not much is known about the structural and dynamical details of its interaction and reactivity with laccases.

Out aim in this work was to study the interaction of this substrate/mediator with a model laccase, CotA from B. subtillis [5], using molecular dynamics simulation studies. In order to address these issues, we parameterized the T1 and T2/T3 centres with quantum mechanical calculations, as well as ABTS in its different oxidation states. Also through these methodolo-gies, we determined the most favorable ABTS conformation in solution. These parameteriza-tions were used to perform molecular dynamics simulations of CotA laccase with ABTS in the different oxidation states, spanning 250 ns for each oxidation state, allowing a good sampling of the conformational space. These simulations elucidated us on the nature of the interaction between the protein and the mediator, and allowed us to identify differences in the free ener-gy of interaction profiles near the T1 centre between the different oxidation states.

Furthermore, the pathways model [6] was used to study the electron transfer properties of the different interaction modes sampled in the molecular dynamics simulations.

[1] E.I. Solomon, U.M. Sundaram, T.E. Machonkin (1996) Chem.Rev. 96, 2563-2605[2] O.V. Morozova, G.P. Shumakovich, S.V. Shleev, Y.I. Yaropolov (2007) Appl. Biochem. Microbiol. 43, 523-535[3] R. Bourbonnais, D. Leech, M.G. Paice (1998) Biochim. Biophys. Acta 1379, 381-390[4] B. Branchi, C. Galli, P. Gentili (2005) Org. Biomol. Chem. 3, 2604-2614[5] I. Bento, L.O. Martins, G.G. Lopes, M.A. Carrondo, P.F. Lindley (2005) Dalton Trans, 3507-3513[6] D.N. Beratan, J.N. Betts, J.N. Onuchic (1991) Science 252, 1285-1288

contactauthor:joãoM.daMas

140

poster 35

Characterization of the terminase complex of phage t7María I. Daudén1, Jaime Martín-Benito1, Rocio Arranz1, Mikel Valle2, Juan Carlos Sánchez1 and Jose L. Carrascosa1

1Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049, Madrid, España. 2CICBiogune, Parque Tecnológico de Bizkaia, 48160, Bizkaia, España

Viral DNA packaging is an attractive system to study the main features of DNA-protein interactions due to the high number of functions and sequential interactions implicated in the packaging itself. Viral packaging of DNA inside preformed proheads is considered the prokariotic equivalent of chromosome condensation, and takes place in a similar way not only in Caudoviridae bacteriopha-ges but also in animal viruses. The structural and functional complexity of phages accomplished by its genetic simplicity make them the perfect model system to study viral packaging [1]. The assem-bly pathway in double stranded DNA (dsDNA) phages, except for some individual features, is highly conserved owing to their common evolutionary origin. The assembly begins with the formation of empty proheads composed by the capsid proteins, the scaffolding proteins and the connector (that links the viral head and the tail). DNA is translocated inside the procapsid passing through the inter-nal channel of the connector with consumption of ATP. This process is performed by the connector and a complex called terminase, which recognizes DNA and presents ATPase and nuclease activity [2]. This complex has been proposed as the macromolecular motor that converts chemical energy from ATP hydrolysis into mechanical movement of DNA inside the procapsid. It is formed by two subunits whose precise stoichiometry has not yet been directly demonstrated.

Our studies focus on the structural and functional characterization of the terminase complex of phage T7. We have cloned the major subunit of the complex (gene product 19) which hydrolyzes ATP and is essential for in vitro packaging assays. We have purified the functional oligomer and its GraFix fixation [3] allowed us to start the structural characterization using electron microscopy and image processing techniques.

As there was no direct evidence of the oligomeric structure of the terminase we started by a 2D image analysis, based on maximum-likelihood multi-reference alignment methods, which clearly shows its 5 fold symmetry, and the subsequent 3D analysis confirmed so. Knowing the symmetry of the complex is essential for studying the influence of matches and missmatches during DNA translocation. Nevertheless the packaging mechanism still remains unraveled and catalytic regions within the terminase structure are not precisely located, so further structural analysis is required. We have design an atomic model of the terminase by threading from already solved atomic struc-ture of a similar protein, to localize the main functional regions inside our EM structure at 17 Å resolution and eventually reveal the existence of several conformational states.

[1] Carrascosa, J. L. V., J.M. (1999) Recent Res. Devel. Virol. 1, 449-465.[2] Cerritelli, M. E., Conway, J. F., Cheng, N., Trus, B. L. & Steven, A. C. (2003) Adv Protein Chem 64, 301-23[3] Kastner, B., Fischer, N., Golas, M. M., Sander, B., Dube, P., Boehringer, D., Hartmuth, K., Deckert, J., Hauer, F., Wolf, E., Uchtenhagen, H., Urlaub, H., Herzog, F., Peters, J. M., Poerschke, D., Luhrmann, R. & Stark, H. (2008) Nat Methods 5, 53-5

contactauthor:MaríaI.daudén

141


poster 36

ph Modulates anionic Currents in pollen protoplastsPedro Dias1,2,3, Patricia Domingos2,3, José Feijó1,2, Ana Bicho2,3.1Departamento de Biologia Vegetal, FCUL, Portugal. 2Grupo de Desenvolvimento Vegetal, IGC, Oeiras, Portugal. 3REQUIMTE, Departamento de Química, FCT-UNL, Caparica, Portugal.

The pollen tube is a cytoplasmic extension which shows one of the fastest polarized cellular growth in nature (up to 4 µm/sec) making this biological system an ideal model for the study of cellular growth and morphogenesis. Previous studies at the I.G.C. have shown by means of ion-specific vibrating probes and intracellular ion imaging for Ca2+, H+ and Cl- that cellular growth control in this biological system is underlined by its ion dynamics. Results demons-trate the occurrence of large chloride fluxes entering the shank of the tube, and its exit at the tip (oscillatory efflux). Additionally, a constitutive alkaline band has been detected at the sub-apical region. [1,2].

Here, we show patch clamp results from pollen protoplasts demonstrating the regulation of chloride channels by pH, an indication of a possible modulator effect in vivo during cellular growth.

Figure 1 - Typical patch clamp chloride whole-cell currents from a pollen grain protoplast of Lilium longiflorum, measured with symmetrical [Cl-] at (A) pH = 5.6 and (B) pH = 6.8. These are characterized by a strong outward rectification and by slow activation and deactivation mechanisms. The change of extracellular pH causes visible alterations on activation and deactivation of the anionic currents (decrease of outward and deactivation currents, increase of inward currents).

[1]Zonia, L., Cordeiro, S., Túpy, J., Feijó, J.A. (2002) The Plant Cell 14, 2233-2249.[2]Holdaway-Clarke, T.L., Hepler, P.K. (2003) New Phytologist 159, 539–563.

contactauthor:pedrodIas

142

poster 37

Modulation of a single copper-ligand bond by remote residues determines the stability of plastocyanins.Estrella Frutos Beltrán1, Sofía Díaz Moreno2, Jesús Chaboy Nalda3, Amin Wagner2, Irene Díaz Moreno1, Francisco Muñoz López1, Simone Raugei4, Paolo Carloni4, Miguel A. De la Rosa1 and Antonio Díaz Quintana1 1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y C.S.I.C.; Avda. Améri-co Vespucio, 49, 41092 Sevilla, Spain. 2Diamond Light Source Ltd. Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX, United Kingdom.3Instituto de Ciencias de los Materiales. C.S.I.C. y Universidad de Zaragoza, 50009 Zaragoza, Spain. 4SISSA/ISAS and INFM-DEMOCRITOS, Modelling Center for Research in Atomistic Simulation, via Beirut 2, 34014 Trieste, Italy.

The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environ-ment is highly conserved in cyanobacterial plastocyanins (Pc). However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis [1,2]. Molecular dynamics computations indicate that differences between the two proteins lie at loop regions located far from the copper site [2,3]. Specific, neutral amino-acid substitutions at this site confer Phormidium Pc a redox-dependent ther-mal stability similar to that of the mesophilic plastocyanin, and vice versa. X-ray absorption spectroscopy of Cu K-edge indicates that the mutation in Phormidium Pc makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis Pc [4]. Analysis of XANES and transitions within the Cu K-edge region of different Pc spectra shows a clear correlation between the thermal stability behaviour of these proteins and the strength of the copper¬-thiolate bond, which is modulated by remote residues outside the barrel [5].

[1] Feio, M.J., Navarro, J.A., Teixeira, M.S., Harrison, D., Karlsson, B.G. and De la Rosa, M.A. (2004) Bio-chemistry 43, 14784–14791.[2] Feio, M.J., Díaz-Quintana, A., Navarro, J.A. and De la Rosa, M.A. (2006) Biochemistry 45, 4900–4906. [3] Muñoz-López, F.J., Raugei, S., De la Rosa, M.A., Díaz-Quintana, A. and Carloni, P. (2010) J. Biol. Inorg. Chem.15, 329–338.[4] Muñoz-López, F.J., Frutos-Beltrán, E., Díaz-Moreno, S., Díaz-Moreno, I., Subías, G., De la Rosa, M.A. and Díaz-Quintana, A. (2010) FEBS Lett. 584, 2346-2350.[5] Chaboy Nalda, J., Díaz-Moreno, S., Díaz-Moreno, I., De la Rosa, M.A. and Díaz-Quintana (2010) sub-mitted.

Work supported by the Andalusian Government (P06 CVI-01713) and the Spanish Ministry of Science and Innovation (BFU2009-07190).

contactauthor:antoniodíazquInTana

143


poster 38

mrna diffusion explains protein gradients in \textit{Drosophila} early development Authors: Rui Dilão and Daniele Muraro

Nonlinear Dynamics Group, Instituto Superior Técnico Av. Rovisco Pais, 1049-001 Lisbon, Portugal

We propose a new model describing the production and the establishment of the stable gradient of the Bicoid protein along the antero-posterior axis of the embryo of Drosophila. In this model, we consider that bicoid mRNA diffuses along the antero-posterior axis of the embryo and the protein is produced in the ribosomes localized near the syncytial nuclei. Bicoid protein stays localized near the syncytial nuclei as observed in experiments.

We calibrate the parameters of the mathematical model with experimental data taken du-ring the cleavage stages 11 to 14 of the developing embryo of Drosophila. We obtain good agreement between the experimental and the model gradients, with relative errors in the range 5-8%. The inferred diffusion coefficient of bicoid mRNA is in the range 4.6x10^{-12}-1.5x10^{-11} m^2s{-1}, in agreement with the theoretical predictions and experimental mea-surements for the diffusion of macromolecules in the cytoplasm. We show that

the model based on the mRNA diffusion hypothesis is consistent with the known observa-tional data, supporting the recent experimental findings of the gradient of bicoid mRNA in Drosophila [Spirov \textit{et al.} (2009) Development 136:605-614].

contactauthor:ruidIlão

144

poster 39

probing the pore drug binding site of microtubules with fluorescent taxanes: evidence of two binding poses.Isabel Barasoain1, Ana M. García-Carril1, Ruth Matesanz1, Giorgio Maccari2, Chiara Trigili1, Mattia Mori2, Jing-Zhe Shi3, Wei-Shuo Fang3, José M. Andreu1, Maurizio Botta2 and J. Fernan-do Díaz1

1Centro de Investigaciones Biológicas. CSIC, Madrid, Spain2Department of Pharmaceutical and Chemical Technology, Faculty of Pharmacy, University of Siena, Italy 3Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing China

The clinical success of taxanes has trigged the search for compounds with a similar mechanism of action but without their inconveniences. This has resulted in the discovery of many compounds with very different chemical structures. These compounds bind to at least three different binding sites. The Laulimalide site[1], the luminal site of taxanes[2], and a site in the external surface of microtubules located in the pore type one,[3] to which taxanes transiently bind in its way to the luminal site, being binding to the luminal and the pore sites mutually exclusive.

Although it is relatively straighforward to measure and model the interactions of the taxanes and taxane-site binding compounds with the luminal binding site[4], little is known about the nature of

the external site, just its location in the type I pore and one of the aminoacids (Thr220) that is labeled by cyclostrep-tin on its way to the inner site[3]. We have kinetically cha-racterized the interaction of taxanes with microtubules employing two fluorescent compounds labeled at diffe-rent positions.

The results indicate that the pore site of microtubule sta-bilizing agents is a new pharmacological target in micro-tubules. Hexaflutax, a drug designed to exclusively bind to the pore site has shown that binding to this site result in cellular effects identical to those produced by classical taxanes, which bind to the luminal site of the microtubule.

The interaction of Hexaflutax with the site has been stu-died using fast kinetic techniques allowing the observa-tion of two different possibilities of interaction of drugs with the pore binding site. Thus the pore site could pro-vide additional binding possibilities to microtubule stabi-

lizing agents, that may justify the apparent promiscuity of the previously thought single paclitaxel site in microtubules.

[1] D. E. Pryor, et al., Biochemistry 2002, 41(29), 9109-9115.[2] E. Nogales, S. G. Wolf, K. H. Downing, Nature 1998, 391(6663), 199-203.[3] R. M. Buey, et al Nat Chem Biol 2007, 3(2), 117-125.[4] R. Matesanz et al, Chem Biol 2008, 15, 573-585.

contactauthor:j.Fernandodíaz

Figure. Docking-based binding modes of Hexaflutax in the binding site.

145


poster 40

role of hydrogen bonds in peptidic systems from coarse-grained simulationsMarta Enciso1, Antonio Rey1

1Departamento de Química Física I. Universidad Complutense de Madrid (Spain)

In this communication we shall discuss the role of backbone hydrogen bonds upon folding/unfolding from a computational approach, paying special attention to the structural and ener-getic properties of secondary structure elements.

Coarse-grained models [1] propound a simplified definition of the system that loses its ato-mistic information but still provides a good description of the protein as a whole. This stra-tegy considerably reduces the computational cost of the calculus, permitting complex simu-lations (e. g. protein folding transitions, wide temperature ranges, great systems sizes...) at reasonable CPU times.

But this approach requires a careful definition of the interacting potential, as huge simplifica-tions are often made. This is especially important in the case of hydrogen bonds, as the cova-lent nature of the interaction plays a key role. For this reason, many hydrogen bond potentials usually obtain abnormal structures and transitions when applied to flexible chain models, and no other geometrical restrictions or energetic contributions are defined into the system.

Here, we present a coarse-grained hydrogen bond potential [2] that successfully reproduces the behavior of backbone hydrogen bonds in flexible systems. We have overcome the pre-viously mentioned obstacles thanks to a detailed treatment of the geometry of the hydrogen bond, based on statistical studies of PDB structures.

For chains of adequate size in a relevant temperature range, we have obtained native-like α-helices and β-sheets in peptidic systems, and reproduced the competition between the populations of these secondary structure elements by the effect of temperature and con-centration changes. Consequently, we present a sketched phase diagram that illustrates the different stability regions in terms of these parameters.

[1] Tozzini, V (2005) Curr. Opin. Struct. Biol., 15, 144-150.[2] Enciso M, Rey A (2010) J. Chem. Phys. 132, 0000

contactauthor:MartaencIso

146

poster 41

a computational study of protein intrafaces and its implication in protein foldingVladimir Espinosa Angarica1,2 and Javier Sancho1,2

1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universi-dad de Zaragoza. Pedro Cerbuna, 12. 50009 Zaragoza, España2Instituto de Biocomputación y Física de Sistemas Complejos, Universidad de Zaragoza. Mariano Esquillor, Edificio I + D, 50018 Zaragoza, España

Despite the large number of studies reported so far regarding protein sequence alignment, functional motif finding, protein-protein and protein-ligand interaction sites, the research of general protein folding mechanisms has not been so deeply addressed. It is known that the tertiary structure of proteins belonging to a given protein family can suffer subtle changes during the course of evolution while the sequence can accommodate a high number of va-riations caused by mutations, insertions and deletions. However, little is known about the conservation of folding pathways in related proteins, and the implications sequence variation could have in the folding process of proteins from the same family. Previous reports have proved that mutations of residues involved in the function, stability, or folding of the protein are less susceptible to be fixed during evolution [1]. Those sequence/structural distinctive fin-gerprints have been widely used to predict functional sites in uncharacterized proteins [2-4]. Nevertheless, most of these works have been focused on the identification of ligand- or pro-tein-protein interaction patches and not on the analysis of structural determinants relevant to folding or stability. In the present work we use combined bioinformatics approaches to try to find out whether proteins from the same family conserve structural features that could deter-mine similar folding mechanisms with other members of the same family distant in sequence and structure with respect to the model protein. The trend of surface fragments to adopt non-native conformations have been published before. However the exact causes leading to this status have not yet being discovered. An earlier work from our group has found an interesting correlation between biophysical properties of the buried interfaces of protein segments and their propensity to adopt unstructured conformations in equilibrium intermediates [5]. Based on these findings we tried to find whether this was a general rule in the protein universe. We analyze a group of protein families belonging to different SCOP classes and folding groups in the search for the conservation of this property of buried interfaces. We found that the polarity ratio of the buried intrafases tend to be conserved in the same region in unaligned 3D structures of members of a protein family. Our results show that protein intrafases and cavities of segments found to be flexible or unstructured in equilibrium intemediates prove to have a different polarity ratio than the rest of the protein. This physical-chemical property is conserved in structural alignments of protein families, which could be related to a conserva-tion of folding pathways in evolutionary related proteins.

1. Orengo CA, Thornton JM: Protein families and their evolution-a structural perspective. Annual review of biochemistry 2005, 74:867-900.2. Jones S, Thornton JM: Prediction of protein-protein interaction sites using patch analysis. Journal of molecular biology 1997, 272(1):133-143.3. Glaser F, Steinberg DM, Vakser IA, Ben-Tal N: Residue frequencies and pairing preferences at protein-protein interfaces. Proteins 2001, 43(2):89-102.4. Pettit FK, Bare E, Tsai A, Bowie JU: HotPatch: a statistical approach to finding biologically relevant features on protein surfaces. Journal of molecular biology 2007, 369(3):863-879.

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5. Campos LA, Bueno M, Lopez-Llano J, Jimenez MA, Sancho J: Structure of stable protein folding in-termediates by equilibrium phi-analysis: the apoflavodoxin thermal intermediate. Journal of molecular biology 2004, 344(1):239-255.

contactauthor:VladimirespInosaangarIca

148

poster 42

Linking Function to Dynamics in globular, Multi-domain and unstructured proteins using nuclear Magnetic resonance SpectroscopySantiago Esteban-Martín1, Robert B. Fenwick1, Xavier Salvatella1,2

1Institute for Research in Biomedicine, Barcelona, Spain. 2Institut Català de Recerca i Estudis Avançats, Barcelona, Spain

A detailed understanding of the biological function of macromolecules requires both knowled-ge of their three dimensional structure and their time-dependent fluctuations. Methods to determine the structure of proteins and nucleic acids are now well established and the static representations that they provide have contributed much to our understanding of protein stability and biological function. The determination of the structural fluctuations at atomic resolution is, by contrast, still in its infancy, particularly for motions taking place at biologi-cally relevant time scales. Recently, however, newly developed methodologies that exploit the information contained in residual dipolar couplings (RDCs) measured using nuclear magnetic resonance (NMR) have provided key insights into the link between the dynamics of macromo-lecules and their biological function. In this communication we present the determination of native ensembles for globular, multi-domain and disordered proteins that explicitly represent their structural heterogeneity in the sub-millisecond time scale. The high resolution descrip-tions of macromolecular dynamics that we have obtained have allowed us to characterize: i) the transfer of structural information across a surface patch in ubiquitin involved in molecular recognition by the proteins that regulate protein degradation, ii) the role of inter-domain mo-tions of T4 Lysozyme in enzymatic catalysis, and iii) the native (residual) contacts in chemica-lly denatured ubiquitin that initiate the folding of this protein [1].

[1] Esteban-Martín S., Fenwick, R.B., and Salvatella, X. (2010) J. Am. Chem. Soc. 132, 4626-4632.

contactauthor:santiagoesTeBan-MarTín

149


poster 43

Computational prediction of single thermal stabilizing mutations in proteins of known structure. Method based on electrostatics, polar accessible surface area and disulfide bridging.Jorge Estrada1,2, Pablo Echenique2,3, Javier Sancho1,2

1Dpto. Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zara-goza, Spain.2Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Universidad de Zaragoza, Spain.3Instituto de Química-Física “Rocasolano”. Consejo Superior de Investigaciones Científicas, Spain.

Predicting stabilizing mutations in proteins is important for practical purposes. We are com-bining several biophysical models to predict stabilizing point mutations. Specifically, our me-thod combines so far three basic models: an electrostatic one, based in the linear Poisson-Boltzmann equation, which allows the study of the contribution of electrostatic interactions to stability; an empirical model based in the polar solvent-accessible surface area of protein residues, specially those in helices; and finally, a geometric model for predicting the creation of disulphide bridges.

This method of prediction of stabilizing mutations in proteins shows the increasing benefits of using computational tools in biophysical studies, and it also shows the possibilities of structu-re-based rational methods both for basic research in biophysics, as well as for its application in industrial environments.

contactauthor:jorgeesTrada

150

poster 44

a mesoscopic model for protein-Dna interaction.R. Tapia-Rojo1,2, D. Prada-Gracia1,2, J. J. Mazo1,3, F. Falo1,2. 1Departamento de Física de la Materia Condensada, Universidad de Zaragoza. 2Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) Universidad de Zaragoza.3Instituto de Ciencia de Materiales de Aragón (ICMA) CSIC-Universidad de Zaragoza.

We propose a simple model for protein-DNA interaccion. DNA description is based on the Peyrard-Bishop-Dauxois model [1] modified to take into account solvation interaction [2]. Protein is modeled by a particle which moves, in 1d, along the DNA chain. Both protein and DNA chain dynamics have been simulated by Langevin molecular dynamics. Analysis of pro-tein trajectory allows us to extract the DNA- protein free energy landscape (FEL) [3]. This method is applied to several promoter genome sequences [4]. We find that basins of the FEL correspond to regulatory and transcription starting sites in promoter sequences.

[1] Dauxois T., Peyrard M, and Bishop AR, (1993) Phys. Rev. E 47, 684[2] Weber G, (2006) Europhysics Letters 73, 806-811 .[3] Prada-Gracia D, Gómez-Gardeñes J, Echenique P and Falo F (2009), PLoS Comput Biol 5(6): e1000415. [4] Alexandrov B.S, V. Gelev, S. W. Yoo, et al. (2009) PLoS Comput Biol 5 e1000313

contactauthor:F.Falo

151


poster 45

Kinetics of the early Stages of transthyretin oligomerization: clues into amyloid formationZaida L. de Almeida1, Tiago Q. Faria1, Rui M. M. Brito1,2

1Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Coimbra, Portugal. 2Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal.

The deposition of insoluble amyloid fibrils is a hallmark of several human pathologies. In the case of Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC) or Senile Sys-temic Amyloidosis (SSA), amyloid fibrils were found to be mainly constituted by Transthyretin (TTR) or its variants [1]. TTR is a 55 kDa tetrameric protein composed by identical subunits of 127 residues which present a two four-stranded β-sheet fold. The β-sheets of two monomers associate to produce a dimer with two eight-stranded β-sheets, and the association of two dimers through hydrophobic interactions yields the functional homotetramer [1]. Conversion of native TTR to amyloid fibrils is a multi-step process initiated by the dissociation of the native protein to non-native monomers which are prone to self-assemble into small soluble oligomers and eventually into amyloid fibrils [2-6]. Increasing interest in the intermediate oligomers arose from several stu-dies showing those as the most cytotoxic species [7,8], thus being a primary target for therapeutic strategies.

In this work, we characterized the kinetics of TTR oligomerization using circular dichroism and in-trinsic tryptophan fluorescence to follow the TTR conformational changes accompanying amyloid formation. In order to separate the initial dissociation process from the oligomerization pathway, TTR assembly was triggered by the addition of salt to acid-unfolded monomers [3,4]. Aiming at a better characterization of the different molecular species in TTR oligomerization pathway, we have used size exclusion chromatography coupled to a multi-angle-laser-light-scattering apparatus. Our data shows that the initial steps of TTR assembly can be described by a two stage process where the first one is concentration-independent while the second process is concentration-dependent. Thus, we propose that the initial stage of amyloid formation from acid-unfolded TTR involves co-llapse of the polypeptide to yield monomeric amyloidogenic intermediates that subsequently self-assemble into small oligomers.

[1] Hamilton, J.A., and Benson, M.D. (2001) Cell. Mol. Life Sci. 58, 1491-1521.[2] Quintas, A., Vaz, D.C., Cardoso, I., Saraiva, M.J.M., and Brito, R.M.M. (2001) J. Biol. Chem. 276, 27207-27213.[3] Lai, Z.H., Colon, W., Kelly, J.W. (1996) Biochemistry 35, 6470-6482.[4] Lindgren, M., Sörgjerd, K., and Hammarström, P. (2005) Biophys. J. 88, 4200-4212.[5] Correia, B.E, Loureiro-Ferreira, N., Rodrigues, J.R., and Brito, R.M.M. (2006) Protein Sci. 15, 28-32.[6] Quintas, A., Saraiva, M.J.M., and Brito, R.M.M. (1999) J. Biol. Chem. 274, 32943-32949.[7] Sousa, M.M., Cardoso, I., Fernandes, R., Guimarães, A., and Saraiva, M.J.M. (2001) Am. J. Pathol. 159, 1993-2000.[8] Sörgjerd, K., Klingstedt, T., Lindgren, M., Kågedal, K., and Hammarström, P. (2008) Biochem. Biophys. Res. Comm. 377, 1072-1078.

contactauthor:q.FarIa

152

poster 46

exploring the folding behavior of peptide dendrimers through computational methodsLuís C. S. Filipe1, Miguel Machuqueiro2, António M. Baptista1

1Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, Avenida da República, EAN, Apartado 127, 2781-901 Oeiras, Portugal; e-mail: [email protected] de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal

Dendrimers are a family of branched compounds that share a common layout where wedges emer-ge radially from a core by means of a regular branching pattern. These structures are characterized by a combination of high end-group functionality and a precisely defined chemical composition. Peptide dendrimers are a specific kind of dendrimers formed by alternating functional amino acids with branching diamino acids [1]. This kind of dendrimers have already rendered models for diffe-rent natural systems; such as catalytic peptide dendrimers, multivalent lectin binding dendrimers and models for vitamin B12 transport proteins [2].

Unfortunately, most studies concerning peptide dendrimers lack structural information at the mo-lecular level. The theoretical study published so far, reported peptide dendrimers presenting sha-pes close to spheres [3], albeit experimental studies on the same systems suggest the existence of more disordered states [2]. Reliable information on the molecular level is therefore vital for un-derstanding how the biological function depends on the biophysical aspects of peptide dendrimers.

Herein we investigate the folding preferences and free energy profiles of five third-generation peptide dendrimers in solution, using long molecular dynamics simulations to sample the confor-mational space of these macromolecules. All the peptide dendrimers studied in the present work share a common topology, and three of them are among the first synthetic models for cobalamin-transport proteins [2].

We explore the radius of gyration of the conformations sampled and, afterwards, by resorting to dissimilarity measures and collective coordinate methods, we construct free energy landscapes and group topographically related molecular geometries. Our studies clearly underline the existence of a myriad of conformational states accessible to these structures; nonetheless, and for the first time, two markedly distinct atomic geometries were identified for peptide dendrimers with common topologies. The differences between these two major conformational forms are patent on their compactness, flexibility and branching distributions.

Our conclusions can be interpreted together with the available experimental results, contributing to a synergistic understanding of the structure-function relation in peptide dendrimers, casting the bases for novel knowledge-based applications.

[1] Darbre, T., Reymond, J.-L. (2006) Acc. Chem. Rev. 39, 925-934.[2] Sommer, P., Uhlich, N.A., Darbre, T., Reymond, J.-L. (2008) ChemBioChem 129, 13238-13246.[3] Javor, S., Reymond, J.-L. (2009) J. Org. Chem. 74, 3665-3674.

contactauthor:luísc.s.FIlIpe

153


poster 47

the specific h-bonding and packing at the interface between theβ-barrel and site 2 are responsible for the different stability of meso- and thermophilic plastocyaninsEstrella Frutos-Beltrán, Miguel A. De la Rosa, Antonio Díaz-Quintana

Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla and CSIC, Sevilla, Spain

The redox state of the copper atom modulates the thermal stability of copper-containing pro-teins, or cupredoxins [1]. Plastocyanin (Pc), in particular, from the mesophilic cyanobacterium Synechocystis sp. (Syn-Pc) and its homologous from thermophilic Phormidium laminosum (Pho-Pc) represent an excellent case of study as their respective thermal stability is affected by the oxidation state of the Cu atom in a different way: Oxidation of the reduced species makes Syn-Pc become more stable but makes Pho-Pc be destabilized [2, 3].

The two proteins show a total sequence identity of 63%, but it drops to 43% when comparing the sequence stretch that form the so-called site 2, or electrostatically charged area. Site 2, which is responsible for the electrostatic interaction of Pc with its physiological partners, shows substantial differences in hydrogen bonding and packing with the β-barrel. Actually, Pho-Pc seems to sacrifice the intermolecular interactions to favour the intramolecular surface salt bridges [4].

In a previous work [5], we designed a neutral point mutation at site 2 that destabilises Pho-Pc and reverses the dependence of its melting temperature (TM) on the redox state. Here, we have carried out several mutations at site 2 of Syn-Pc to test their effect on both protein sta-bility and its dependence on the oxidation state of copper. Some of the mutations located at the L5 loop raises the TM value of oxidised Syn-Pc, while the substitution of Gly60 by alanine induces both an increase in the TM value of the oxidised species and a decrease in TM of the reduced form.

1. Wittung - Stafshede, P. Inorg. Chem 2004, 43, 7926-33. 2. Feio, M. J., Navarro, J. A., Teixeira, M. S., Harrison, D., Karlsson, B. G., De la Rosa, M. A. Biochemistry 2004, 43, 14784-91. 3. Feio, M. J., Díaz-Quintana, A., Navarro, J. A., De la Rosa, M. A. Biochemistry 2006, 45, 4900-6. 4. Muñoz-López, F. J., Raugei, S., De la Rosa, M. A., Díaz-Quintana, A., Carloni, P. J. Biol. Inorg. Chem. 2010, 15, 329-38. 5. Muñoz-López, F.J., Frutos-Beltrán, E., Díaz-Moreno, S., Díaz-Moreno, I., Subías, G., De la Rosa, M.A., Díaz-Quintana, A. FEBS Lett. 2010, 548, 2346-50.

contactauthor:estrellaFruTos-BelTrán

154

poster 48

the Functional and Structural role of Lys377 in the Secondary transporter Melibiose permease from e. coliOliver Fuerst1,Yi-Bin Lin1, Meritxell Granell1, Gérard Leblanc2, Víctor A. Lórenz-Fonfría1 and Esteve Padrós1

1Centre d’Estudis en Biofísica, Depart. de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain 2Laboratoire de Physiologie des Membranes Cellulaires-LRC-CEA 16V, Université de Nice Sophia-Antipolis and CNRS (UMR 6078), 06238, Villefranche sur Mer, France

The melibiose transporter from Escherichia coli (MelB) is a 12 α-helical membrane protein in charge of the accumulation of α-and β-galactosides, e.g., the sugar melibiose [1]. MelB couples the unfavorable transport of the sugar into the cell with the favorable co-transport of a cation (H+, Na+ or Li+) down its electrochemical gradient, in a 1:1 stoichiometry.

To understand how MelB functions on a molecular level, it is indispensable to indentify essen-tial residues, as those involved in substrate binding. Evidence for key residues often comes from mutants with impaired or altered substrate transport, while the identification of their specific role requires further biochemical/biophysical characterization. Lys377 in the putati-ve helix XI of MelB is an illustrative example. Mutation of Lys377 to Cys, Val or Asp impairs sugar transport in cells [2]. But whether Lys377 is a functional or a structural key residue for MelB has not been addressed yet.

In this contribution, we characterized several mutants of Lys377 by substrate-induced Fourier-transform Infrared (FTIR) and fluorescence spectroscopies, which report substrate binding by their effect on the protein conformation [3]. In this way we studied the ability of K377C, K377V and K377C/I22S mutants to bind melibiose and Na+. Our results point out that, in contrast to the C-less (a WT-like form devoid of cysteines), all three Lys377 mutants lack any response to melibiose, suggesting a complete loss of the sugar binding site. Likewise, these mutants also lacked any response to Na+, with the exception of the double-mutant K377C/I22S. This double mutant retained Na+ binding, although with reduced affinity, lea-ding to spectral/conformational changes in the protein differing from the ones seen in the C-less MelB.

The second derivative of the IR absorbance spectra for K377C and K377V showed repro-ducible changes in the amide I band with respect the C-less. Since the amide I is sensitive to the protein backbone conformation it indicates that these two mutants have an altered MelB basal structure. In contrast, the amide I band of the double mutant K377C/I22S showed much reduced changes compared to the WT, indicated a preserved native structure, which explains the partially restored Na+ binding observed in this double mutant, and supports the essential role of Lys377 for melibiose binding.

From our present data we can conclude that Lys377 has a direct functional role in MelB, be-ing essential for melibiose binding, and a possible important but not essential amino acid for Na+ binding. Additionally, Lys377 plays also a key structural role in preserving MelB native structure, presumably by the formation of a salt bridge.

[1] Leblanc,G., Pourcher,T., & Zani,M.L. (1993) Soc. Gen. Physiol Ser. 48, 213-227.[2] Franco,P.J., Jena,A.B., & Wilson,T.H. (2001) Biochimica et Biophysica Acta (BBA) - Biomembranes 1510, 231-242.

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[3] León,X., Lórenz-Fonfría,V.A., Lemonnier,R., Leblanc,G., & Padrós,E. (2005) Biochemistry 44, 3506-3514.

contactauthor:oliverFuersT

156

poster 49

testing the mechanical hypothesis in scaffoldins: the effect of natural linkers and cellulase bindingAlbert Galera-Prat, Andrés Manuel Vera, Alejandro Valbuena, Javier Oroz & Mariano Carrión-Vázquez

Instituto Cajal, CSIC, CIBERNED & IMDEA Nanociencia, Avda. Doctor Arce 37, 28002 Madrid email: [email protected]

Cellulosomes are large protein complexes capable of degrading cellulose in a highly efficient manner1. In Clostridium thermocellum and C. cellulolyticum, scaffoldin, which is the largest and non-catalytical component of the cellulosome, is a modular protein composed of different modules. A CBM (“carbohydrate binding module”) and a type II dockerin module allow the bacteria to attach the whole complex to its substrate (cellulose) and to the cell itself, respec-tively; cohesin I modules bind dockerin I bearing enzymes1. This complex, as a cell adhesion system it is expected to be subjected to mechanical stress2. In a previous study, we hypothe-sized that cohesin I modules located between the two anchoring points of scaffoldin might be exposed to mechanical stress and hence those would have higher mechanical stability than those outside this region (“hanging” modules). In that study, we showed that cohesin modules putatively subjected to mechanical stress are the most mechanostable proteins reported to date, while “hanging” cohesins present lower stability3. In the present study we have analy-zed by AFM-SMFS (atomic force microscopy-based single-molecule force spectroscopy) and MD (molecular dynamics) simulations the mechanical stability of different cohesin modules located between and outside the anchoring points. These results give further support to this hypothesis. Furthermore, we have studied the effect on the mechanical stability of different elements naturally found in the cellulosome. These include the natural surrounding linkers between cohesin I modules and the bound cellulases, both of which may possibly affect the mechanical stability of cohesins4,5,6. Taken together, our results reinforce our current mo-del: the mechanical clamp of scaffoldin cohesins is the major molecular determinant of the mechanical barrier that protects the integrity of this module and in turn its binding surface to cellulases.

[1] Bayer, EA., Belaich, JP., Shoham, Y, Lamed, R. (2004) Annu. Rev. Microbol. 58:521-54.[2] Bustamante C., Chemla YR., Forde NR., Izhaky D. (2004) Annu. Rev. Biochem. 73:705-48.[3] Valbuena A., Oroz J., Hervás R., Vera AM., Rodríguez D., Menéndez M., Sulkowska JI., Cieplak M., Carrión-Vázquez M. (2009) Proc. Natl. Acad. Sci. 18:13791-96.[4] Cao Y., Yoo T., Zhuang S., Li H. (2008) J. Mol. Biol. 378:1132-41.[5] Politou AS., Gautel M., Joseph C., Pastore A. (1994) FEBS letters. 352:27-31.[6] Rounsevell RW, Steward A, Clarke J. (2005) Biophys. J. 88:2022-9.

contactauthor:albertgalera-praT

157


poster 50

Skl and pal: so different but so similarCristina Gallego1,2, José Luis Sáiz1,2, Fernando Díaz3, Laura Lagartera1,2, Pedro García2,3, Marga-rita Menéndez1,2 1Instituto de Química-Física “Rocasolano”, CSIC, Serrano 119, 28006 Madrid2Ciber de Enfermedades Respiratorias (CIBERES)3Centro de Investigaciones Biológicas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid

Skl and Pal are the endolysins encoded by bacteriophages SK137 and Dp-1, respectively. Both have N-acetylmuramoyl-L-alanine amidase activity and hydrolyze the cell wall of Strep-tococcus pneumoniae causing the lysis and the death of the bacteria, hence their interest as possible antimicrobials (enzybiotics) in the treatment of diseases caused by this human pa-thogen of the Firmicutes phylum. Both amidases have a modular organization; they comprise a catalytic module located at the N-terminus, and a cell-wall binding module (CBM) located at the C-terminus that recognizes the choline residues present in pneumococcal teichoic and lipoteichoic acids. The CBM of Skl and Pal, made up of 6 tandemly-arranged repeats of 17-23 amino acids and a C-terminal segment of 15 residues, is essential for the lytic activity on intact cell-walls and is also involved in the choline-dependent self-association equilibria dis-played by different CBPs (Choline Binding Proteins) [1]. Pal catalytic module belongs to the Amidase-5 family (PF05382) which has been related to the NlpC/P60 family of amidases and peptidases [2], recently included in the CHAP (Cysteine Histidine Amidohydrolase/Peptidase) superfamily [3], to whom the catalytic module of Skl belongs (PF05257). All of them are in-cluded in the peptidases CA clan (CL0125).

The structural and thermodynamic characterization of Skl and Pal has revealed important similarities between their catalytic modules despite the low identity (~8%) shown by their sequences. They include the module folding, a catalytic triad comprising cystein, histidine and a polar residue, and probably the same reaction mechanism. In contrast, the CBMs exhibit remarkable differences in their affinity for choline, although they share 51% of their sequen-ces (72% similarity) and type of folding. Besides, the aromatic residues involved in choline recognition are fully sequence conserved in both endolysins. Finally, the specific features of Skl and Pal have been analyzed trying to further understand of the phenomena influencing the activities of both enzymes, and their potential relevance to other pneumococcal cell-wall hydrolases.

[1] Varea et al., (2004) J. Biol. Chem. 275, [2] Anantharaman, A. & Aravind, L. (2003). Genome Biology 4:R11 [3] Layec et al. (2008). J Mol Microbiol Biotechnol. 14: 31-40

contactauthor:cristinagallego

158

poster 51

Modulation of Functional properties of Cytochrome c by phosphorylation José M. García-Heredia, Maria Salzano, Antonio Díaz-Quintana, Miguel A. De la Rosa, Irene Díaz-Moreno

Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC

Respiratory Cytochrome c (Cc) is a well-known protein that plays a crucial role in cell life (respiration) and death (apoptosis). Such a double physiological function is regulated by tyro-sine nitration and phosphorylation - both occurring inside the mitochondria. The number of tyrosine residues of Cc ranges from four (horse) to five (human) and six (plant). Four of them are highly conserved: Tyr67 (internal), Tyr48 (intermediate), Tyr74 and Tyr97 (solvent acces-sible) [1, 2]. In vivo phosphorylation of Tyr48 or Tyr97, in particular, impairs the function of Cc as electron carrier in respiration [3, 4].

Thus we have designed two phospho-mimic mutants of human Cc - in which Tyr48 and Tyr97 have been specifically replaced by glutamate - to get a further insight into the physi-co-chemical and functional properties of phosphorylated Cc. The Y97E mutation makes the melting temperature (Tm) of Cc drastically decrease from 85.9 ºC to 59.0 ºC, but the Y48E substitution hardly affects the Tm of Cc (Tm, 84.7 ºC). However, the latter mutation shifts the pKa value of the alkaline transition from 9.5 to 7.0, a finding that could have a great bio-logical relevance. Moreover, the Apaf-1-mediated activation of caspase-9 in vitro induced by the Y48E mutant decreases two fold in comparison to either the wild–type or Y97E species.

[1] Rodríguez-Roldán, V., García-Heredia, J.M., Navarro, .JA., Hervás, M., De la Cerda, B., Molina-Heredia, F.P., De la Rosa, M.A. (2006) Biochem. Biophys. Res. Commun. 346, 1108-1113.[2] García-Heredia, J.M., Díaz-Moreno, I., Nieto, P.M., Orzáez, M., Kocanis, S., Teixeira, M., Pérez-Payá, E., Díaz-Quintana, A., De la Rosa, M.A. (2010) Biochim. Biophys. Acta – Bioenergetics (in press).[3] Lee, I., Salomon, A.R., Yu, K., Doan, J.W., Grossman, L.I., Hüttemann, M. (2006) Biochemistry 45, 9121-9128.[4] Yu, H., Lee, I., Salomon, A.R., Yu, K., Hüttemann, M. (2008) Biochim. Biophys. Acta 1777, 1066-1071.

contactauthor:joséM.garcía-heredIa

159


poster 52

the nMr structure of the n-terminal domain of tubulin Cofactor C reveals a disordered fragment involved in tubulin bindingMaría Flor García-Mayoral1, Raquel Castaño2, Juan Carlos Zabala2, Manuel Rico1, Marta Bruix1.1Departamento de Química Física Biológica, Instituto de Química Física Rocasolano, CSIC, Madrid, Spain.2Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria-IFIMAV, Santander, Spain.

Human Tubulin Cofactor C (TBCC) is a two-domain 346-aminoacid protein involved in the post-chaperonin folding pathway of α- and β-tubulin monomers. In association with other cofactors, TBCC forms a supercomplex in the ultimate folding steps inducing the GTP hydro-lysis that leads to the release of productive tubulin heterodimers ready to polymerize into microtubules [1, 2].

We have used NMR spectroscopy to determine the 3D structure of the N-terminal domain of TBCC. The domain adopts a spectrin-like fold topology composed of an antiparalell three-helix bundle connected by short loops. Remarkably, our NMR data reveal that the 30-residue N-terminal fragment of the domain remains flexible and disordered in solution, although several regions retain significant helical propensities.

We show that the N-terminal domain of TBCC interacts with the αβ-tubulin dimer and that the N-terminal 30-residue fragment actively participates in the binding event. This fragment concentrates mostly charged residues, leading to the proposal of a likely electrostatically-driven molecular recognition process involving the acidic hypervariable regions of tubulin monomers.

[1] Fontalba, A., Paciucci, R., Avila, J., and Zabala, J.C. (1993) J Cell Sci. 106, 627-632.[2] Tian, G., Huang, Y., Rommelaere, H., Vandekerckhove, J., Ampe, C., and Cowan, N.J. (1996) Cell 86, 287-296.

contactauthor:MaríaFlorgarcía-Mayoral

160

poster 53

eM Characterization of a Clostridium thermocellum Mini-Cellulosome Begoña García-Alvarez1, Fernando M. V. Dias3, José A. M. Prates3, Carlos M. G. A. Fontes3, Maria João Romão2, Ana Luísa Carvalho2 and Oscar Llorca1 1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Rami-ro de Maeztu, 9, 28040 Madrid, Spain.2REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universi-dade Nova de Lisboa, 2829-516 Caparica, Portugal.3Centro Interdísciplinar de Investigação em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Rua Professor Cid dos Santos, 1300-477 Lisboa, Portugal.

The plant cell wall degrading enzymes of most anaerobic bacteria are organized in the form of a large supra-molecular complex, named the “cellulosome”, containing a protein scaffold that holds an extensive repertoire of glycoside hydrolases [1, 2]. In the cellulosome of the anaerobic bac-terium Clostridium thermocellum, the enzymes are bound to a non-catalytic protein termed the “scaffoldin” (CipA), which, in turn, binds to cell-surface anchoring proteins [3]. CipA contains nine reiterated cohesin domains [4] which interact with the corresponding dockerin domains located in cellulosomal enzymes [5].

A C. thermocellum mini-cellulosome comprising three Cel8A cellulases bound to a three cohesin domain (C3-C4-C5) scaffoldin that derives from CipA was produced. The mini-cellulosome was observed at the electron microscope and single-molecule images have been used to derive 2D averages of the data with a higher signal-to-noise ratio. These 2D analyses provided information about the overall architecture of the cellulosome sub-complex. Our main efforts aimed at determi-ning if the cellulosome constitutes a unique 3D structure, a range of defined and limited 3D con-formations, or a highly flexible complex. The preliminary analysis of the mini-cellulosome images obtained in the electron microscope suggested that only a limited range of possible conformations of the cellulosome are observed. If this is finally proved to be correct, single molecule images of the protein sub-complex will be used to define the 3D arrangement of the mini-cellulosome. This structure will be interpreted with the help of the fitting of structures of isolated domains solved at atomic resolution using X-ray crystallography into the EM map using computational approaches, to derive a pseudo-atomic model of CipA.

[1] Shoham, Y., Lamed, R. and Bayer, E. A. (1999) Trends Microbiol, 7, 275-281.[2] Béguin, P. and Aubert, J. P. (1994) FEMS Microbiol. Rev. 13, 25-58. [3] Béguin, P. and Lemaire, M. (1996) Crit. Rev. Biochem. Mol. Biol. 31, 201-236.[4] Gerngross, U. T., Romaniec, M. P., Kobayashi, T., Huskisson, N. S. and Demain, A. L. (1993) Mol. Mi-crobiol. 8, 325-334.[5] Salamitou, S., Raynaund, O., Lemaire, M., Coughlan, M., Béguin, P., and Aubert, J. P. (1994) J. Bacteriol. 176, 2822-2827.

Acknowledgements: The authors would like to acknowledge the Portuguese Fundação para a Ciência e a Tecnologia for financial support through project PTDC/QUI-BIQ/100359/2008

contactauthor:Begoñagarcía-alVarez

161


poster 54

Folding of prp and the Species BarrierLeszek A. Gierusz1, David S. Pearson2, Michael A. Geeves2 and Teresa J. T. Pinheiro1

1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. 2De-partment of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.

Transmissible spongiform encephalopathies, which include bovine spongiform encephalopa-thy in cattle, scrapie in sheep and Creutzfeldt-Jakob disease (CJD) in humans, are associated with the conversion of the normal cellular form of the prion protein (PrPC) to an altered pa-thological form, often referred to as the scrapie isoform (PrPSc).

According to the “protein-only” hypothesis, the PrPSc is capable of self-replication upon asso-ciating with the cellular form and converting it to pathological isoform via template-assisted mechanism (1, 2). Although the details of prion conversion are not fully understood, it is generally accepted that this transformation involves refolding of PrP from its cellular form to an altered disease-associated isoform.

An important phenomenon known as the species barrier affects prion transmission, resulting in significantly longer incubation time and lower incidence of disease upon its transfer bet-ween different species.

Another feature of prion disease is disease-modulating polymorphisms in the PrP amino acid sequence which can alter individuals` susceptibility to infection. One of such polymorphisms is the Q167R mutation which confers increased disease resistance.

Those phenomena are linked to the primary sequence of PrP, and thus comparing the folding properties of the prion protein from different species provides valuable insight into the me-chanism of species barrier and disease susceptibility.

Here we examined the folding properties of PrP from two different species and the mutant Q167R associated with increased disease resistance. Dramatic differences in the mechanism of folding of these proteins contribute to our understanding of the mechanisms of the species barrier and susceptibility to prion disease.

(1) Bolton D.C., McKinley M.P., Prusiner S.B. (1982) Science 218, 1309–1311.(2) Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95, 13363–13383.

contactauthor:leszeka.gIerusz

162

poster 55

a tailor-made optical tweezers to study the dynamics of the addaB helicase-nucleaseBenjamin Gollnick1, Joseph T. P. Yeeles2, Mark S. Dillingham2 and Fernando Moreno-Herrero1

1Department of Macromolecular Structures, Centro Nacional de Biotecnología, CSIC, Cam-pus de Cantoblanco, Madrid, Spain.2Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, UK.

Double-stranded DNA breaks are a frequently occurring and potentially catastrophic form of DNA damage that can lead to cell death, premature ageing or cancer. In bacteria, recombi-national repair is initiated by a class of enzymes called helicase-nucleases. The prototypical member of this class in the model organism Bacillus subtilis is the protein complex AddAB. It processes a DNA end to a 3’-terminated single-stranded DNA overhang, thus creating a subs-trate for the subsequent steps of recombinational repair [1, 2].

To study the role of force in the complex process of DNA repair initiation, we have built a tailor-made single-beam Optical Tweezers based on a published design [3]. Our setup includes both commercial and custom-designed components. It combines a high-power infrared laser with a single high-numerical-aperture microscope objective, providing a stable optical trap in three dimensions. Position detection of trapped microspheres is done by high-speed video microscopy with a bandwidth of up to 0.4 kHz. Initial calibration measurements suggest a spatial resolution of around 5 nm, depending on the applied laser power, and an effective overall force range of 0.2 – 80 pN. Our novel instrumentation will provide us with the means to characterize and monitor the real-time dynamics of DNA repair processes catalyzed by the molecular motor AddAB. So far, preliminary pulling experiments using a glass micropipette as a fixed anchor point are ongoing. Next, we aim to be able to measure the stalling force of AddAB. These experiments and future developments of the Optical Tweezers device will be discussed in detail.

[1] Chedin, F., Ehrlich, S.D. and Kowalczykowski, S.C. (2000) J. Mol. Biol. 298, 7-20.[2] Yeeles, J.T.P. and Dillingham, M.S. (2007) J. Mol. Biol. 371, 66-78.[3] Keyser, U.F., Van der Does, C., Dekker, C., Dekker, N.H. (2006) Rev. Sci. Instrum. 77, 105105.

contactauthor:BenjamingollnIcK

163


poster 56

Characterization of the effect of curcumin on the catalytic activity of pKCα. Gómez-Fernández, J.C., Pérez-Lara, A. and Corbalán-García, S.

Dpto. Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, 30100-Murcia, Spain.

Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in several functions such as cell differentation, metabolism, growth and apoptosis. PKC isoforms play an important role in several diseases being a subject of research and drug development, par-ticularly in cancer research. Curcumin, a polyphenol, is the active principle of the rhizomes of the plant Curcuma longa. Curcumin possess many pharmacological applications acting as anti-infective, anti-tumoral and anti-oxidant agent.

This work aims to assess the effect of curcumin on the activity of PKCα and its relation with different enzyme’s effectors such as phosphatidylserine (POPS), Ca2+, dioleylglycerol (DOG) and PIP2. We also studied the physico-chemical properties of membranes containing curcu-min and its possible interaction with the C1 and C2 regulatory domains. For that purpose, we used mutants abolishing the function of the C1 (W58G) or the C2 (D246-248N) domain of PKCα and we found a complex effect on both domains that is also influenced by the physical properties of curcumin-containing membranes.

contactauthor:j.c.góMez-Fernández

164

poster 57

Structure of the n-terminal region of the plakin domain of Bp230 and mapping of the integrin α6β4 binding siteMaría Gómez Hernández1, Arnoud Sonnenberg2, José M de Pereda1

1Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas – Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain.2Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Hemidesmosomes (HDs) are multiprotein complexes that mediate firm adhesion of epithelial cells to the underlying basement membrane [1]. The 230-kDa bullous pemphigoid autoan-tigen (BP230/BPAG1) and the integrin α6β4 (a laminin receptor) are components of the HDs. BP230 belongs to the plakin family of cytolinkers that associate with and cross-link components of the various cytoskeletal systems. BP230 contains in its N-terminal region a plakin domain (a sequence of ~ 1000 residues conserved in the plakin family) formed by eight Spectrin Repeats (SR2 to SR9) and a SH3 domain inserted in the SR5. The N-terminal region of BP230 is important for its recruitment into HDs as it associates with the cytoplasmic region of the β4 subunit of the integrin α6β4 [2].

We have designed a collection of BP230 fragments corresponding to the SR2 and the 55-resi-dues long N-terminal tail that precedes the SR2, and shorter versions that contain N-terminal deletions up to the beginning of the SR2. The recombinant proteins were expressed in E coli. Here we report the crystal structure of the SR2 of BP230. The structure has been solved by molecular replacement and was refined against data to 2.0 Å resolution. The SR2 structure is built up of three α-helices connected by short loops. Comparative analysis by circular di-chroism of the SR2 and longer fragments that contain increasing sequences of the N-terminal tail suggests that the tail is an intrinsically disordered region.

It has previously been shown by yeast two hybrid experiments that binding of BP230 to integrin β4 relies on the sequence 1-98 [3]. In order to determine the affinity for β4 of the different fragments of BP230 we have developed an assay based on measuring changes in the fluorescence anisotropy of Oregon Green-labeled BP230 proteins. By using this assay we have narrowed down the β4-binding site to a 18-residue long sequence in the N-terminal tail of BP230.

[1] Litjens, S.H., J.M. de Pereda, and A. Sonnenberg, (2006) Trends Cell Biol,. 16(7): p. 376-83.[2] Jefferson, J.J., et al. (2006) J Mol Biol, 11: p. 11.[3] Koster, J., et al. (2003) J. Cell Sci, 116(Pt 2): p. 387-99.

contactauthor:MaríagóMezhernández

165


poster 58

a mechanical model of the tip link suggests its role in acoustic mechanotransductionÀngel Gómez-Sicilia, Luis Laurence-Martínez, Javier Oroz, Alejandro Valbuena & Mariano Carrión-Vázquez

Instituto Cajal, CSIC, CIBERNED & IMDEA Nanociencia, Avda. Doctor Arce 37, 28002 Madrid e-mail: [email protected]

In the process of audition, hair cells in the inner ear play a crucial role1. These cells convert sound waves into electrical impulses through a mechanotransduction machinery. An impor-tant component of this machinery is the tip link2, which joins and coordinates the movement of these cilia. Recently, it has been discovered that such connectors are composed by two members of the cadherin superfamily: cadherin 23 and protocadherin 153,4. The function of the tip link is still a matter of discussion: hypotheses span from considering it as the gating spring of the system to regarding it as a rigid force transmitter. The aim of this study is to mo-del the mechanical properties of the tip link, based on the helical geometry of the cuaternary structure of cadherin 235, and to verify the results of this model experimentally. The results obtained should suggest the specific mechanical role of this complex in the auditory system.

1. Hudspeth AJ: How the ear's works work. Nature 1989, 384:397-404 2. Fettiplace R, Hackney CM: The sensory and motor roles of auditory hair cells. Nature Rev Neurosci 2006, 7:19-29. 3. Kazmierczak P et al: Cadherin 23 and protocadherin 15 interact to to form tip-link filaments in sen-sory hair cells. Nature 2007, 449:87-92. 4. Müller U: Cadherins and mechanotransduction by hair cells. Curr Opin Cell Biol 2008, 20:557-66. 5. Tsuprun V et al: The structure of tip links an kinocilial links in avian sensory hair bundles. Biophys J 2004, 87:4106-12.

contactauthor:ÀngelgóMez-sIcIlIa

166

poster 59

Dielectric constant of planar supported biomembranes measured at the nanoscaleLaura Fumagalli1,2 Aurora Dols1,2 Georg Gramse1,2 Daniel Esteban1 Martin Edwards1 Gabriel Gomila1,2

1Nanobioelec Group, Institut of Bioengineering of Catalonia (IBEC), Barcelona (Spain). 2Departament of Electronics, University of Barcelona, Barcelona (Spain)

The dielectric constant of membranes is an important parameter of cell bioelectricity as it quanti-fies the intrinsic dielectric behaviour of the plasma membrane at low-frequencies (< 1MHz) in pro-cesses such as membrane potential formation, action potential propagation or ion membrane trans-port. Furthermore, it determines the cell response to externally applied electrical fields employed by bioelectrical techniques such as dielectrophoresis, impedance spectroscopy or electroporation.

Existing experimental techniques provide low-frequency dielectric constant values for cell membra-nes with spatial resolution limited, in the best of the cases, to a few microns. However, probing the dielectric constant at the nanometer scale is still of fundamental interest because of the rich local structure of membranes and the fact that many bioelectric phenomena occur at the nanoscale. In the present communication we report on recent results obtained by our group concerning the use of electric scanning probe techniques for the quantitative extraction of the low-frequency dielec-tric constant of biomembranes at the nanoscale. We will describe two technical implementations for this purpose, namely, one based on local capacitance measurements [2], [3] (Fig. 1, left) and a second one based on local electrostatic force measurements [4] (Fig. 1, right). Essential aspects re-lated to sample preparation, system callibration and theoretical modeling will be provided. Results obtained on supported purple membrane monolayers and lipid bilayers will be presented.

Fig. 1: Nanoscale mapping of the dielectric constant of a single purple membrane patch obtained from local capacitance [2] (left) and electrostatic force [3] (right) measurements.

[1] Kalinin, S. and Gruverman, A. eds., (2007) Scanning Probe Microscopy, electrical and electromecha-nical phenomena at the nanoscale (New York, Springer, 2007).[2] Fumagalli, L., Ferrari, G., Sampietro, M. and Gomila,G. (2009) Nano Lett. 9, 1604.[3] Casuso, I., Fumagalli, L., Gomila,G., Padrós, E., (2007) Appl. Phys. Lett. 91, 063111.[4] Gramse,G., Casuso, I., Toset, J. and Gomila, G. (2009) Nanotechnology 20, 395702.

contactauthor:gabrielgoMIla

167


poster 60

Catalase-like activity of human met-hemoglobin: a mechanistic and kinetic study.María I. González-Sánchez1, Francisco García-Carmona2 and Edelmira Valero1.1Department of Physical Chemistry. Industrial Engineering School. University of Castilla-La Mancha. Albacete, Spain.2Department of Biochemistry and Molecular Biology A. Biology Faculty. University of Murcia. Murcia, Spain

Hemoglobin is a member of the hemoprotein superfamily whose main role is to transport O2 in vertebrate organisms. In addition it shows peroxidase activity, being able to catalyze the one-electron oxidation of a great variety of substrates in the presence of H2O2. In the absence of these agents, H2O2 acts both as oxidizing and reducing substrate for hemoglobin, being decomposed to molecular oxygen in a similar way to the action of classical catalases. In the present work, the production of oxygen by methemoglobin in the presence of H2O2 was kinetically characterized with a Clark-type electrode. Values of initial velocity show a linear phase of approximately 0.5-1.0 min. Kinetic data reveal non-michaelian behaviour with Hill coefficients in the range 1.2-1.6. It is not the first time that deviations of Michaelian beha-viour are shown by hemoglobin [1]. The effect of pH was also studied: catalase-like activity was pH dependent and favoured under neutral conditions. To obtain more information about the mechanism, superoxide radical scavengers such as superoxide dismutase, tetranitrome-thane and manganous ions were added to a medium containing methemoglobin and H2O2. In summary results shown can be explained by means of a suicide substrate (H2O2)-based mechanism that includes three H2O2-consuming routes of Hb: the catalase-like pathway, the peroxidase-like pathway and the inactivation pathway. The system evolves in transient phase until there is a total loss of activity, never reaching a steady-state. The number of turnovers (r) of H2O2 required to completely inactivate haemoglobin was found to be 61.7 ± 0.6. We have proposed that the main protective mechanism of metHb towards oxidative damage of H2O2 is the catalase-like process. The resolution of the differential equations set corresponding to the mechanism leads to obtaining the equation for the time-dependence of oxygen evolution during the inactivation of metHb in the presence of a large excess of H2O2. The results here shown could contribute to the knowledge of the mechanism of methemoglobin under oxida-tive stress conditions, where it is considered a potentially toxic molecule when released from erythrocytes during hemolysis, inflammation, or tissue injury.

[1] González-Sánchez, M. I., Manjabacas, M. C., García-Carmona, F. and Valero, E. (2009). Chem. Res. Toxicol. 22, 1841-1850.AcknowledgmentsThis work was supported by Projects PAI08-0175-8618 and POII10-0235-8597 from the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha (JCCM, Spain).

contactauthor:MaríaI.gonzález-sánchez.

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poster 61

Direct action in potassium channels of kidney cells by diuretic drugs and aldosterone Patrícia Gonçalves1, Terese Moura1, Ana Isabel Santos2, Ana Bicho1.1REQUIMTE – Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Univer-sidade Nova de Lisboa, 2829-516 Caparica, Portugal.2Departamento de Fisiologia, Faculdade de Ciências Médicas, FCM, Universidade Nova de Lisboa, 1169-056 Lisboa, Portugal.

Hypertension represents the major cause of death and incapacity in developed countries. Diuretic drugs, together with a low salt diet, are clinically used to control hypertension by elevating the rate of urine excretion and decreasing the extracellular volume. These drugs modulate the sodium and potassium transport, subsequently followed by water movements. The mechanism by which diuretics may act on potassium channels is not completely understood [1]. In the present study, HEK293 kidney cells were used as a model to study the effect of two diuretic drugs, caffeine and acetazolamide (diamox, an inhibitor of the carbonic anhydrase), on the potassium passive currents. In addition, aldosterone (hormone that regulates Na+ and K+ transport by the cortical collecting duct) was also studied.

Two endogenous potassium currents from HEK293 cells [2, 3] were studied under whole-cell voltage-clamp conditions using the patch clamp technique [4]. Two channels showing positive cu-rrents and different voltage dependencies were observed. Depolarizing voltage steps from -20mV to +90mV triggered an outward rectifier persistent current, while depolarizing voltage steps from -80mV to +90mV additionally triggered fast inactivating currents (similar to neuronal A type cu-rrents). The two diuretics had different effects on the currents. Caffeine (1mM) had an inhibitory effect on the two types of currents, while acetazolamide only inhibited the outward persistent current. The effect of aldosterone was obtained by i) direct application of 1mM in the bath solution and ii) overnight (300µM aldosterone). The two tests results show distinct inhibitory effects. When applied directly in the bath solution aldosterone inhibited the fast inactivating currents. HEK293 cells grown in the presence of 300µM aldosterone did not show a component of the whole cell cu-rrent due, showing a possible regulatory effect of the hormone on DNA transcription for a specific channel population. Our data suggest a possible direct mechanism of regulation of kidney potas-sium channels by diuretic drugs and the hormone aldosterone. Further experiments are needed in order to understand the mechanisms by which these drugs modulate the potassium channels from HEK293.

[1] Carrageta, M. (2007). Acedido em: 5 de Maio de 2007, em: http://cardiologia.browser.pt/Primeira-Pagina.aspx.[2] Yu, S. P., and Kerchner, G. A. (1998) Journal of Neuroscience Research 52, 612-617. [3] Zhu, G., Zhang, Y., Xu, H., & Jiang, C. (1998) Journal of Neuroscience Methods 81, 73-83.[4] Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflügers Arch. Eur. J. Physiol. 391:85-100.

contactauthor:patríciagonçalVes

169


poster 62

implication of the trp domain of trpv1 in functional couplingLucia Gregorio-Teruel1, Pierluigi Valente1 and Antonio Ferrer-Montiel1. 1Instituto de Biología Molecular y Celular. Universidad Miguel Hernández. 03202 Elche. SPAIN.

TRPV1 (Transient receptor potential vanilloid 1) is a member of the TRP channel family activated by physical and chemical stimuli. TRPV1 functions as a non selective tetrameric cation channel with high permeability to calcium ions. Each subunit shows a topology of six α-helical transmembrane segments with a pore region between the fifth and sixth seg-ment. The cytoplasmatic N- and C-termini contains several aminoacid residues involved in the modulation of channel gating. Specifically, the C-terminal region contains the TRP domain (Glu684-Arg721), a highly conserved sequence in the TRP channels family. This region is a molecular determinant in the functional coupling of the channel. To further understand the role of this region in the protein functionality we performed a site directed mutagenesis strategy on a non functional TRPV1 chimera, TRPV1-AD2, that contains the TRP domain of TRPV2. Complementary, we studied the role of residues I696, W697 and R701 in the functional coupling of TRPV1. To perform these experiments we replaced each position by the all natural aminoacids. We characterize the expression level and the electrophysiological properties of the different mutants obtained for both strategies. Taking together, our data suggest that the TRP domain region is critical for the functional coupling of the activating stimuli. Particularly, we found that mutations of this region affected the energetic of channels opening. These results demonstrate that the preservation of these positions is essential for the correct functionality of TRPV1.

Funded by MICINN, CONSOLIDER-INGENIO 2010, ISCIII, Fundació La Marató de TV3

- García-Sanz et al. (2004) J. Neurosci., 24, 5307-5314

- García-Sanz et al. (2007) J. Neurosci., 27, 11641-11650

- Valente et al. (2008).FASEB J. 9, 3298-309

contactauthor:luciagregorIo-Teruel

170

poster 63

Biophysics of protein misfolding in metabolic disease: Defective folding and function on etF mutantsBárbara J. Henriques1, Rikke K. Olsen2, Peter Bross2, Cláudio M. Gomes1,*1Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal2Research Unit for Molecular Medicine, Århus University Hospital, Skejby, Århus, Denmark

Mutations in the ETFA/ETFB genes cause multiple acyl-CoA dehydrogenase deficiency (MADD), a congenital metabolic disease with a broad clinical expression. These mutations result in defective or absent electron transfer flavoprotein (ETF), a key enzyme from the mitochondrial beta oxidation pathway. Known mutations fall essentially in two groups: mutations affecting protein folding and assembly, and mutations impairing catalytic activity and interactions with partner dehydrogenases. We have analysed three of these mutations, ETFβ C42R, D128N and R191C which typify different scenarios in respect to the clinical phenotypes of the patients in which they were identified. The ETFβ-C42R mutation, associated with a severe MADD phenotype, impairs protein folding. Upon co-expression with GroEL and DnaKJ, in an E. coli system, an increase in soluble protein production is noted, although no enzymatic activity was restored, probably as a defective insertion of the AMP and FAD cofactors.

Proteins harboring the two other mutations, ETFβ-D128N and ETFβ-R191C, associated with mild MADD, were purified in soluble form after heterologous expression. Mutations leading to mild cli-nical phenotypes, are frequently associated with poor catalytic activity of the mutant variant, and deficient FAD insertion, and in these cases some patients respond dramatically to pharmacologic doses of riboflavin (1,2). We have used a combination of biophysical and biochemical methods to address the impact of mutations on ETF folding, conformational stability and tightness in me-diating electron transfer. The two mutant variants present the same overall α/β fold topology of native ETF, but both have substantially decreased enzymatic activity and conformational stability. We have also found that cofactor insertion in vitro substantially improves the folding and stability of mutants, and that the presence of flavin prevented proteolytic digestion by avoiding protein destabilization (3). These results show how FAD can work as a pharmacological chaperone, and the mechanism through which riboflavin supplementation in patients can inhibit aggregated/mis-folded protein accumulation. Altogether, these studies correlate available clinical and cellular data with a detailed molecular understanding of the structural and functional basis of MADD pathology, contributing to define structural hotspots within the ETF fold that are useful to rationalize the effects of MADD mutations.

1. Gianazza, E., Vergani, L., Wait, R., Brizio, C., Brambilla, D., Begum, S., Giancaspero, T. A., Conserva, F., Eberini, I., Bufano, D., Angelini, C., Pegoraro, E., Tramontano, A., and Barile, M. (2006) Electrophoresis 27(5-6), 1182-11982. Yotsumoto, Y., Hasegawa, Y., Fukuda, S., Kobayashi, H., Endo, M., Fukao, T., and Yamaguchi, S. (2008) Mol Genet Metab 94(1), 61-673. Henriques, B. J., Rodrigues, J. V., Olsen, R. K., Bross, P., and Gomes, C. M. (2009) J Biol Chem 284(7), 4222-4229

contactauthor:Bárbaraj.henrIques

171


poster 64

Structural rearrangements in the kinase module of FaD synthetase during catalysisBeatriz Herguedas1, Juan A. Hermoso2, Marta Martínez-Júlvez1, Milagros Medina1 1Departmento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and BIFI, Universidad de Zaragoza, E-50009 Zaragoza, Spain.2Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, C.S.I.C. Serrano 119, 28006-Madrid, Spain

Riboflavin is converted in FMN and FAD in two sequential reactions catalyzed by Riboflavin kinase (RFK) and FMN Adenylyltransferase (FMNAT) activities. Prokaryotes usually contain a bifunctional enzyme that catalyzes both reactions, the FAD synthetase (FADS), whereas eukaryotes contain two monofunctional enzymes each one responsible for one of the activi-ties1. The crystal structure of the bifunctional enzyme from Corynebacterium ammoniage-nes (CaFADS) revealed the folding in two modules, RFK and FMNAT, as well as trimeric and hexameric assemblies in which RFK and FMNAT modules from different protomers approach their catalytic sites2. Here, we present the crystal structure of the individually-expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP. The structure was solved by Molecular Replacement and the model was refined to 1.65 Å resolution. Flavin and adenine binding to the RFK module, a small β-barrel of about 15 kDa, induces impor-tant structural changes, but maintains the conformation of the loops involved in the interac-tion with the FMNAT module. The structure of the complex shows structural rearrangements which close the flavin binding site and open the adenine cavity, whereas in the CaFADS apo form these sites were opened and closed, respectively. Residues far from the catalytic site in the apo-structure are displaced about 15 Å towards the catalytic site to interact with FMN and ADP, suggesting a role in the reorganization of the protein or in catalysis. All these observa-tions are in agreement with the ligand-binding studies3, which indicated that ADP only binds to the RFK module when FMN is present. These data additionally provide a structural-based mechanism for FMN biosynthesis in prokaryotes.

[1] Frago, S., Serrano, A., Martínez-Júlvez, M., and Medina, M. (2008). BMC Microbiol. 8, 160.[2] Herguedas, B., Martínez-Júlvez, M., Frago, S., Medina,M., and Hermoso, J.A. (2010). J. Mol. Biol. In press.[3] Frago, S., Velázquez-Campoy, A., and Medina, M. (2009). J. Biol. Chem. 284, 6610–6619.

contactauthor:Beatrizherguedas

172

poster 65

reconstitution of bacterial cell division proteins onto phospholipid bilayer nanodiscsVíctor M. Hérnandez-Rocamora1, Germán Rivas1

1Departamento de Biología Físico-Química, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, E-mail: [email protected]

Phospholipid bilayer nanodiscs are novel well-defined model membranes derived from high-density lipoprotein particles that have proven useful to maintain membrane proteins in a native-like monodisperse bilayer, while simultaneously in solution (Bayburt and Sligar, 2009. FEBS letters in press DOI: 10.1016/j.febslet.2009.10.024). They enable the performance of biochemical and biophysical assays to study membrane protein behavior and interactions by means of techniques such as analytical ultracentrifugation, light scattering and fluorescence spectroscopy (Alami et al., 2007. EMBO J. 26:1995-2004).

We are interested in the reconstitution of the multi-protein complex that initiates cell divi-sion in E. coli (the proto-ring) onto biomimetic membranes (including nanodiscs). The proto-ring complex is formed by three proteins (the GTPase FtsZ- bacterial ancestors of tubulin, FtsA – an amphitropic protein, ands ZipA – a membrane-bound protein) that assemble at the cytoplasmic membrane forming a structure required for the incorporation of the remaining division proteins (Vicente et al., 2006. J. Bacteriol. 188, 19-27).

Here we report the incorporation of ZipA into phospholipid bilayer nanodics and the prelimi-nary interaction experiments with FtsZ, as an example of the usage of this technology to study protein-protein interactions with membrane proteins.

contactauthor:VíctorM.hérnandez-rocaMora

173


poster 66

triptophan mutations in hirsutellin a produce a non-cytotoxic but ribonucleolitically active ribotoxinElías Herrero-Galán1,2, Javier Lacadena3, Álvaro Martínez del Pozo3, Nieves Olmo3, Mercedes Oñaderra3 and José G. Gavilanes3.1Instituto Madrileño de Estudios Avanzados IMDEA-Nanociencia, Madrid, Spain.2Centro Nacional de Biotecnología del Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.3Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.

Ribotoxins are a family of fungal extracellular ribonucleases that are able to interact with membranes, enter cells and impair ribosome function by specifically cleaving a single bond in the sarcin-ricin loop (SRL) [1, 2]. Hirsutellin A (HtA) has been recently described as the smallest member of this group known to date, as its 130 aminoacids reproduce all the abili-ties of previously described ribotoxins (149/150 aa long), among which α-sarcin is the best characterized member [3, 4]. In this work we have explored the residues involved in both the ribonucleolytic activity of HtA and its membrane interacting interfaces. By site directed mu-tagenesis we have found that an Asp residue, located in the position equivalent to Tyr48 of α-sarcin, plays an important role in the active site of HtA, what is a novelty in the ribotoxins family. Furthermore, we have studied the implication of tryptophan residues in the membra-ne interaction ability of HtA, finding that W71 and W78 in loop 2 of HtA are important for entering the cell. Additionally, activity assays with mutants affecting this residues as well as W65 have shown that hydrophobic interactions with additional regions of the ribosome in the proximity of the SRL are also involved in substrate recognition. Finally, the variant W65/71/78F has resulted to be the first non-cytotoxic but ribonucleolitically active ribotoxin variant produced to date.

[1] Lacadena, J., Álvarez-García, E., Carreras-Sangrà, N., Herrero-Galán, E., Alegre-Cebollada, J., García-Ortega, L., Oñaderra, M., Gavilanes, J.G., and Martínez del Pozo, A. (2007) FEMS Microbiol. Rev. 31, 212-237.[2] Herrero-Galán E, Álvarez-García E, Carreras-Sangrà N, Lacadena J, Alegre-Cebollada J, Martínez del Pozo A, Oñaderra M, Gavilanes JG (2009) in Microbial Toxins: Current Research And Future Trends (Edited by T Proft), pp. 167-187. Caister Academic press, Auckland, New Zealand.[3] Herrero-Galán, E., Lacadena, J., Martínez del Pozo, A., Boucias, D.G., Olmo, N., Oñaderra, M., and Gavi-lanes, J.G. (2008) Proteins 72, 217-228. [4] Viegas, A., Herrero-Galán, E., Oñaderra, M., Macedo, A.L., and Bruix, M. (2009) FEBS Journal 276, 2381-2390.

contactauthor:elíasherrero-galán

174

poster 67

Single-molecule characterization of the mechanical properties of dsrna by means of optical-tweezersElías Herrero-Galán1,2, José María Valpuesta2, José L. Carrascosa2 and José Ricardo Arias-González1,2

1Instituto Madrileño de Estudios Avanzados IMDEA-Nanociencia, Madrid, Spain.2Centro Nacional de Biotecnología del Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

The development of single-molecule techniques has allowed in recent years the mechani-cal characterization of individual DNA molecules, shedding light on their elastic properties and the physical constraints that affect the proteins that interact with them [1], apart from enabling the use of DNA for nanotechnological purposes [2]. Over the past few years, dsRNA has emerged as a far more important ingredient in the life cycle of a cell than previously ex-pected, but the intrinsic difficulty of working with RNA has hindered the development of new tools to study this molecule. We have engineered long dsRNA molecules for their individual characterization by means of optical tweezers [3,4]. By studying their mechanical properties and comparing them with those of DNA, we aim both to provide a new tool for potential na-notechnological applications and to gain new insight into the basic features of dsRNA-protein interactions. Here, we present the first complete force-extension curves for dsRNA reaching the overstretching transition together with calorimetric data and discuss the results in com-parison with the elastic and stability parameters of DNA.

[1] Bustamante, C., Bryant, Z., and Smith S.B. (2003) Nature 421, 423-427.[2] Seeman, N.C. (2003) Nature 421, 427-431.[3] Smith, S.B., Cui, Y., and Bustamante, C. (2003) Meth. Enzimol. 361, 134-162.[4] Hormeño, S., and Arias-González, J.R. (2006) Biol. Cell 98, 679-695.

contactauthor:elíasherrero-galán

175


poster 68

Mechanical Stability of Low-humidity Single Dna Molecules with polycationsSilvia Hormeño1,2, Fernando Moreno-Herrero2, Borja Ibarra1,2, José L. Carrascosa2, José María Valpuesta2 and J. Ricardo Arias-González1,2. 1Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia). Canto-blanco, 28049 Madrid, Spain2Department of Macromolecular Structure, Centro Nacional de Biotecnología, CSIC. Calle Darwin nº 3. Cantoblanco, 28049, Madrid, España

Recent years have witnessed the emergence of an entirely new field of science, often refe-rred to as single-molecule biophysics. This interdisciplinary field is currently providing novel approaches to biochemical and biological problems [1]. The capability of single-molecule methods of revealing the behavior of individual molecules having different conformation and properties is beyond the ability of their bulk counterparts, which describe the behavior of enormous ensembles of molecules, averaging the measured parameters over the entire mo-lecular population.

Optical tweezers are one of these new techniques which allow the real-time manipulation of single molecules and cells [2]. They have been used in a variety of biological systems to lead to a new understanding of the mechanical properties of the fundamental building blocks of the cell, and of the mechanism by which molecular machines function. We have used this techni-que to study the mechanical stability of single DNA molecules under low humidity conditions. Besides simulating dehydration conditions which affect DNA in vivo these experiments give us information about two important biological processes: condensation and the B-A confor-mational transition of DNA at the single molecule level. We have analyzed the simultaneous effect of tension and ethanol on two DNAs differing in both sequence and secondary structure in buffer to analyze GC content dependencies. Our results support that condensation co-exists with the A-form and that such association reinforce the conformational B-A transition. The study is complemented with the characterization by two other single-molecule techniques, namely, magnetic tweezers and atomic force microscopy.

[1] Bustamante, C., Macosko, J. C., and Wuite, G. J. (2000) Nat. Rev. Mol. Cell. Biol. 1, 130-136.[2] Hormeño, S., and Arias-González, J. R. (2006) Biol. Cell 98, 679-695.

contactauthor:silviahorMeño

176

poster 69

Fusion peptide effects on epitope recognition at membrane surfaces by the broadly neutralizing anti-hiV-1 2F5 monoclonal antibodyNerea Huarte1, Aitziber Araujo1, Rocío Arranz2, José M. Valpuesta2 and José L. Nieva1

1Biophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular Biology Department, University of the Basque Country, PO Box 644, 48080 Bilbao, Spain. 2Department of Macromolecular Structures, National Center for Biotechnology (CSIC), Canto-blanco, 28049 Madrid, Spain.

The HIV-1 glycoprotein fusogenic subunit gp41 is the target for 2F5, a broadly neutralizing monoclonal antibody (MAb2F5) isolated from asymptomatic infected individuals. The 2F5 epitope locates close to the membrane interface within the membrane proximal external re-gion (MPER) that connects the HIV-1 envelope gp41 ectodomain with the transmembrane anchor. Here evidence is presented indicating that the conserved amino-terminal fusion pep-tide (FP) increases the affinity of this antibody for its membrane-inserted epitope. Structural characterization by circular dichroism together with membrane-disrupting activity measure-ments suggests the formation of FP-MPER complexes at the surface of lipid bilayer vesicles. MAb2F5 associated more efficiently with lipid vesicles containing FP and MPER peptides as compared to those containing only MPER, or MPER in combination with FPctl, a scrambled version of the FP. Moreover, the N-terminal FP sequence had almost no effect on membrane-inserted epitope binding by MAb4E10, a neutralizing antibody that has been shown to extract its C-terminal MPER epitope from the membrane interface. In combination with recently re-ported crystallographic data [1], these results support a “catch-and-hold” mechanism for the process of MAb2F5-epitope binding at membrane surfaces.

[1] Julien JP, Bryson S, Nieva JL, Pai EF (2008) J. Mol. Biol., 384, 377-392

contactauthor:nereahuarTe

177


poster 70

the phi29 Dna polymerase presents an active Dna unwinding mechanismBorja Ibarra1, Jose A. Morín1, Ricardo J. Arias-González1, José M. Lázaro2, Margarita Salas2, José M. Valpuesta3, José L. Carrascosa3

1IMDEA Nanociencia, Facultad de Ciencias, Universidad Autónoma Madrid, 28049, Madrid, Spain.2Centro de Biología Molecular “Severo Ochoa” (CBMSO-CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain. 3Centro Nacional de Biotecnología (CNB-CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain.

During DNA replication mechanical unwinding of the DNA helix is required for the advance of the replication machinery. Unlike many DNA polymerases the bacteriophage Phi29 DNA poly-merase presents a ‘helicase-like’ activity and is able to couple DNA replication and unwinding within the same polypeptide. Using Optical Tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of DNA unwinding by the Phi29 DNA polymerase as the protein replicates processively the DNA. A DNA hairpin is hold between an optical trap and a mobile surface. As a single polymerase works on the hairpin its replication and unwinding activities can be measured in real time (by measuring the change in extension in the DNA polymer), revealing the fluctuations of their rates in response to the DNA sequen-ce and force applied in the direction of unwinding. The sequence and force sensitivity of the unwinding reactions of the wild type and an unwinding-deficient polymerase mutant are not consistent with a passive unwinding mechanism or active unwinding mechanisms already proposed for specialized helicases (like T7 and NS3). Our results indicate that the Phi29 DNA polymerase presents its own active unwinding mechanism that may substantially differ from the unwinding mechanism used by specialized nucleic acid helicases.

contactauthor:BorjaIBarra

178

poster 71

exploring the Structural and energetic Determinants of Ligand Binding to transthyretin: Searching guidelines for the rational Design of therapeutic Drugs against ttr amyloidosis Catarina S. H. Jesus1,2, Cláudia V. S. Moniz1, Pedro Cruz1, Adrian Velazquez-Campoy3 and Rui M. M. Brito1,2

1Chemistry Department, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal. 2Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.3Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Universidad de Zaragoza, Zaragoza, Spain.

Amyloid fibril formation and deposition have been associated with a wide range of diseases, inclu-ding spongiform encephalopathies, Alzheimer’s disease, Parkinson’s disease, type II diabetes, fami-lial amyloid polyneuropathies (FAP), and senile systemic amyloidosis (SSA), among many others. In pathologies such as SSA and certain forms of FAP, the amyloid fibrils are mostly constituted by transthyretin (TTR) and variants of TTR, a homotetrameric plasma protein implicated in the trans-port of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, which was first isolated from a Portuguese kindred. L55P-TTR is one of the variants associated with the most aggressive form of FAP. Until now, the only known therapeutic approach to TTR amyloidosis is liver transplantation. New therapeutic strategies are being developed taking advantage of our current understanding of the mechanism of TTR amyloidosis [1]. Two main ideas are being explo-red: i) stabilization of the native tetrameric form of TTR; and ii) solubilization of amyloid fibrils and pre-fibrillar aggregates [2].

As an integral part of a high throughput virtual screening campaign to find new leads for TTR amyloid inhibitors, we set out to characterize in detail the structural and energetic determinants of known TTR binders. Of all techniques currently available to characterize protein-ligand binding, isothermal titration calorimetry (ITC) is the only one capable of measuring not only the magnitude of the binding affinity but also the magnitude of the the enthalpic (ΔH) and entropic (ΔS) contribu-tions to ligand-binding. Because the enthalpic and entropic contributions are related to structural parameters, they can be used as a guide for the rational design of potential lead compounds and for validation of computational predictions of binding energetics. Additionally, studies based on Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) experiments allow fast identification of the ligands that specifically bind to a protein and may reveal the ligand interaction site at atomic level.

Here, we will report ITC data to characterize the thermodynamic binding profiles of four ligands to WT-TTR: thyroxine (T4), diethylstilbestrol (DES), flufenamic acid (FF) and a polychlorinated biphen-yl compound (PCB). Additionally, using STD-NMR we characterize and identify the ligand groups that specifically interact with the the binding pockets of WT-TTR.

[1] Quintas, A., Vaz, D. C., Cardoso, I., Saraiva, M. J. M., and Brito, R. M. M. (2001) J. Biol. Chem., 276, 27207-27213.[2] Brito, R. M. M., Damas, A. M., and Saraiva, M. J. (2003) Curr. Med. Chem. Immun. Endoc. Metab. Agents 3, 349-360.

contactauthor:catarinas.h.jesus

179


poster 72

reconstitution of e. coli proto-ring complex inside giant unilamelar vesicles made from bacterial inner membranes Ariadna Martos1, Miguel Vicente2, Germán Rivas-Caballero1* and Mercedes Jiménez1*1Chemical and Physical Biology Programme, Centro de Investigaciones Biológicas, CSIC, Madrid2Centro Nacional de Biotecnología, CSIC, Madrid* Corresponding authors: [email protected] (GRC) and [email protected] (MJ)

The activity, the interactions and polymerization properties of FtsZ are being measured using topologically restricted reconstructions of the proto-ring in artificial and natural membranes structured as giant unilamelar vesicles (GUVs). Artificial membranes are from E. coli. polar lipid extracts and natural membranes are available from E. coli. cytoplasmic membrane. Being more complex than artificial lipid vesicles, natural membranes maintain a composition and distribution of proteins and lipids resembling more closely the native membrane. To provide a native cell environment for these assays, we formulate a cytomimetic medium that reprodu-ces the crowded intracellular environment, physiological osmolarity and energy supply pools. This set of tools will contribute to define the physicochemical conditions that modulate the energetics and dynamics of FtsZ association to the cytoplasmic membrane and its eventual dissociation from it.

contactauthor:MercedesjIMénez.

180

poster 73

Sphingosine incorporation into negatively-charged lipid bilayers modifies their structural propertiesNoemi Jiménez- Rojo, Ana R. Viguera, Félix M. Goñi, Alicia Alonso

Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain.

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the basic building block of sphin-golipids. In the last decade it has been shown to act as a potent metabolic signaling molecule, by activating a number of protein kinases. The present contribution intends to describe some physical properties of sphingosine in lipid bilayers. Sphingosine increases the permeability of phospholipid bilayers, giving rise to vesicle leakage. This effect is attributed to a peculiar property of sphingosine when interacting with certain lipid mixtures, namely the stabilization of high-melting domains. Since at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g. in bilayers containing phosphati-dic acid together with phosphatidylethanolamine and cholesterol) gives rise to a fast release of vesicular contents. Moreover, sphingosine appears to be sensitive to lipid oxidation: only when bilayer lipids are partially oxidized does sphingosine elicit vesicle aggregation.

contactauthor:noemijIMénez-rojo.

181


poster 74

reconstitution of proapoptotic bak function in liposomes reveals a dual role for mitochondrial lipids in the membrane permeabilization processOlatz Landeta, Ane Landajuela and Gorka Basañez

Unidad de Biofisica, Centro Mixto CSIC-UPV/EHU, Barrio Sarriena s/n, Leioa 48940 (SPAIN).

BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP), whose detailed mechanism of action in intact cells remains ill defined mainly due to the inherent complexity of the intracellular apoptotic machinery. Here, we demonstrate, for the first time that basic aspects of BAK proapoptotic function can be reproduced in a cell-free system con-sisting of recombinant BAK and tBID proteins combined with synthetic vesicles emulating the lipid composition of the mitochondrial outer membrane. Using this minimalistic in vitro reconstituted system we obtained direct experimental proof that BAK possesses an intrinsic pore-forming activity which can be unleashed by tBID. The BAK -driven membrane permeabi-lization process triggered by tBID is associated with membrane insertion and oligomerization of the protein, and culminates with formation of lipidic-toroidal pores. We also identified a dual role for selected mitochondrial lipids along the molecular pathway of BAK -induced membrane permeabilization. First, cardiolipin specifically and directly interacts with BAK lea-ding to a localized structural rearrangement in the protein, which shares defined features with a conformational change observed in endogenous mitochondrial BAK early during apoptosis. Second, several lines of evidence indicate that LPC and DAG, two curvature-inducing lipids present in mitochondrial membranes, modulate the energetic expenditure required to form lipidic-toroidal pores by BAK. Collectively, these results strongly support the notion that during apoptosis BAK functions as a direct effector of MOMP akin to BAX, and also provide important mechanistic insights into potential roles played by specific mitochondrial lipids within the BCL-2 regulated MOMP pathway.

contactauthor:olatzlandeTa

182

poster 75

Modulation of the FMn electronic properties by the protein environment of anabaena FlavodoxinIsaias Lans, Susana Frago, and Milagros Medina

Departament of Biochemistry and Molecular and Cellular Biology and Institute of Biocompu-tation and Physics of Complex Systems (BIFI). Universidad de Zaragoza. Zaragoza. Spain.

Flavodoxin (Fld) from Anabaena is a protein that under iron-deficient condition replaces fe-rredoxin as electron carrier from photosystem I to Ferredoxin-NADP+ reductase. Fld presents as cofactor non–covalently FMN molecule bound to the apoprotein. The flavin ring of FMN can exist in three different redox states (oxidized, semiquinone or hydroquinone) within the protein environment, since upon interaction of FMN with the apoprotein the Eox/sq and Esq/hq values for the bound FMN result inverted and well-separated. These three different redox states are characterised by a different number of electrons and protons in the flavin ring. To investigate the influence of the protein environment on the electronic properties of the different positions of the FMN isoalloxazine ring QM/M molecular dynamic simulations of Fld reconstituted with five different FMN analogs were carried out using the AMBER and Gaussian program. The obtained models in oxidized, semiquinone and hydroquinone states, and electronic properties were used to predict and calculate the electrons entry and exit sites within the flavin ring. According Fukui function the entry and exit of electrons will take pla-ce over N5 and C4a atom of isoalloxazine ring in the Fld oxidized and hydroquinone states, respectively, for all the reconstituted Flds. The same position expected for all the free FMN analogs here studied. However, in the Fld semiquinone state differences in the positions for the entrance of electrons can be predicted between the full protein and the free semiquinone FMN analogues.

contactauthor:Isaiaslans

183


poster 76

When topology is not enough: evaluating a new coarse-grained hybrid potential for protein foldingMaría Larriva* and Antonio Rey

Departamento de Química Física I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, [email protected]*

Molecular simulation is a powerful tool to study how a polypeptide chain acquires its charac-teristic and functional three-dimensional structure in aqueous solution. In fact, coarse-grained representations, combined with efficient sampling algorithms (Monte Carlo simulations), can yield a global view of this process with a manageable computational cost.

Topology-based potentials have been successfully used to characterize the folding process in proteins with lower level of frustration. Despite this fact, there are many others in which these reduced potentials do not work properly. We have developed a new hybrid potential including the amino acid sequence: In this model, two beads per residue are required to represent the protein and the energy function is both topology and sequence-dependent.

In order to study the sequence effects on the folding pathways, we have tackled the folding process of the ribosomal protein S6 from Thermus thermophilus (S6T) and 5 circular permu-tants (P13-14, P33-34, P54-55, P68-69 and P81-82) [1] by 2 different simulation models: a topological-based model and our hybrid model, including explicit sequence information in the energy function.

In this communication, we present some results that show how the sequence contribution modifies the folding properties. Finally, we have compared the simulation data for the hybrid model with experimental results [1]. The agreement between experimental and simulation results (at least at a qualitative level) suggests that, in this case, our hybrid model is more appropriate to describe the transition than the topological one, highlighting the crucial role of the sequence in the folding process.

[1] Haglund E., Lindberg M.O., and Oliveberg M. (2008) J. Biol. Chem. 283, 27904-27915.

contactauthor:MaríalarrIVa

184

poster 77

exploring the conformational space of pyruvate carboxylase by Cryo-eM, new insights into its mechanism of actionGorka Lasso1, Linda P.C. Yu2, David Gil1, Song Xiang2, Liang Tong2, Mikel Valle1,3

1Structural Biology Unit, Center for Cooperative Research in Biosciences bioGUNE, 48160 Derio, Spain2Department of Biological Sciences, Columbia University, New York, NY 10027, USA3Department of Biochemistry and Molecular Biology. University of the Basque Country. P.O. Box 644, 48080 Bilbao, Spain

Pyruvate carboxylase (PC) is a conserved multifunctional enzyme (EC 6.4.1.1) that carboxyla-tes pyruvate into oxaloacetate in two sequential chemical reactions. This enzyme plays a crucial role in the intermediate metabolism and it is linked to important diseases such as lactic acidaemia and type-2 diabetes. PC crystal structures show four monomers arranged in two la-yers with two monomers per layer [1-3]. Despite recent progress, further work is still needed to understand the complex mechanism of action of this multifunctional enzyme.

Cryo-electron microscopy (cryo-EM), maximum-likelihood based classification and both ma-nual and flexible fitting methods were used to explore the conformational variability of PC in the presence of different ligands. All density maps show noticeable conformational diffe-rences between layers, mainly for the biotin-carboxyl carrier protein (BCCP) domain and the biotin carboxylase (BC) domain. The results demonstrate that the BCCP domain localizes near the BC domain of its own monomer and travels to the carboxyltransferase (CT) domain of the opposite monomer within the same layer. A conformational change is observed for the PC tetramerization (PT) domain, suggesting a new functional role in communication among mo-nomers. A long-range communication pathway between subunits located in different layers, via their interacting PT-PT and BC-BC domains, may be responsible for the cooperativity of PC from S. aureus.

1. Xiang, S., and Tong L. (2008) Nat Struct Mol Biol. 15(3), 295-302.2. St Maurice, M., Reinhardt, L., Surinya, K.H., Attwood, P.V., Wallace, J.C., Cleland, W.W., and Rayment, I. (2007) Science 317(5841), 1076-1079.3. Yu, L.P., Xiang, S., Lasso, G., Gil, D., Valle M., and Tong, L. (2009) Structure 17(6), 823-832.

contactauthor:gorkalasso

185


poster 78

a revised Model for rnase a oligomerization via 3D Domain Swapping Based on the Biophysical Characterization of its Conformational ensemble in 40% acetic acidJorge Pedro López Alonso1, Marta Bruix1, Josep Font2, Marc Ribó2, Maria Vilanova2, María Ángeles Jiménez1, Jorge Santoro1, Carlos González1, Douglas Laurents1.1Instituto de Química Física “Rocasolano” C.S.I.C. Serrano 119 Madrid 28006 Spain. 2Laboratori d’Enginyeria de Proteïnes, Universitat de Girona, Girona 17071, Spain.

RNase A is a normally monomeric protein which can form several oligomers by 3D domain swap-ping of its N-terminal α-helix, C-terminal β-strand or both [1-3]. Treatment with 40% acetic acid induces RNase A oligomerization, and has long been thought [1-3] to act by locally unfolding hinge loops to detach the terminal segments and thereby facilitate swapping between monomers once the acid is removed by lyophilization and the protein is returned to an aqueous solution at neutral pH. Using several biophysical techniques, the conformation of RNase A in 40% acetic acid and 8 M urea (pH 2.5) has been characterized. RNase A is thoroughly unfolded in 8 M urea at pH 2.5. It is chiefly unfolded in 40% acetic acid; while the native helical structure is partly retained, the β-sheet is fully unfolded, tertiary contacts between nonpolar groups are lost and all X-Pro peptide bonds are mostly in the trans conformation. Kinetic experiments show that refolding from the denatured ensemble produced by 40% acetic acid occurs via an intermediate with native-like structure but non-native trans X-Pro peptide bonds. This same intermediate is populated during refolding from concentrated solutions of GdmCl [4], and we find that urea- or GdmCl-denatured RNase A can also oligomerize during refolding. These findings lead us to propose a revised model for RNase A oligomerization via 3D domain swapping. Since 3D domain swapping is a probable mechanism for amyloidogenesis [2,5], these finding have implications for amyloid formation.

[1] Crestfield, A.M., Stein, W.H. & Moore, S. (1962) ABB Suppl. 1, 217.[2] Liu, Y., Gotte, G., Libonati, M., & Eisenberg, D. (2001) Nat. Struct. Biol. 8, 211-4.[3] Gotte, G., Laurents, D. & Libonati, M. (2006) Biochem. Biophys. Acta 1764, 44-54.[4] Laurents, D., Bruix, M., Jamin, M. & Baldwin, R.L. (1998) J.M.B. 283, 669-78.[5] Sambashivan, S., Liu, Y., Sawaya, M. R., Gingery, M. & Eisenberg, D. (2005) Nature, 437, 266–269.

contactauthor:douglaslaurenTs

186

poster 79

Comparative study of nucleophosmin and nucleoplasmin: two not so similar proteins.Benoit Lectez1, Jorge Falces1, Igor Arregi1, Sonia Bañuelos1, Petr Konarev2, Dmitri Svergun2, Stefka Taneva1 and María A. Urbaneja1.1Unidad de Biofísica (CSIC/UPV-EHU), Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Bilbao, Spain.2European Molecular Biology Laboratory, Hamburg outstation c/o DESY, Hamburg, Germany.

Nucleophosmin (NPM1) is a ubiquitously expressed protein and one of the most represented nu-cleolar proteins [1]. NPM1 is a key component of several cellular processes as ribosome biogenesis, control of centrosome duplication, maintenance of genomic stability and cell cycle regulation. The protein exerts these functions thanks to its interaction with various partners and to its constant shuttling ability from the nucleus to the cytoplasm and backward [1]. To perform this traffic, which depends on the cell cycle, NPM1 displays import, export and nucleolus localization signals. NPM1 consists of several domains: the N-terminal region, named “core”, is structurally homologous to that of nucleoplasmin (NP) and presents a β-structure responsible of the oligomeric structure [2, 3]. The C-terminal domain, of ca. 50 residues and missing in NP, has an autonomously folded α-helical structure [4]. Since both NP and NPM1 proteins belong to the same histone chaperone family, we have undertaken a comparative study of structural and functional properties of these two proteins. Hence, we have characterized the solution structure of full-length NPM1 by small angle X-ray scattering. Ab initio and rigid body modeling allow us to reconstruct a pentameric particle at low resolution. The overlap of this model with that of NP shows that both proteins have similar overall shapes, with the exception of NPM1 C-terminal domains which are perceived as globular bodies protruding from and connected through the linker to the pentameric core. This C-terminal part is a frequent target of oncogenic mutations in acute myeloid leukemia. Mutations-induced changes at the C-terminal domain compromise its stability and cause aberrant translocation of the protein to the cytosol. Regarding to these data, we have compared the structural stability of the full-length NPM1, the N-t and C-t isolated domains with that of NP and NP core domain [5, 6]. We have also characterized the binding properties of NPM1 to the nucleus-cytoplasm transport receptors.

[1] Grisendi S, Mecucci C, Falini B, Pandolfi PP. (2006) Nature Reviews 6, 493-505.[2] Namboodiri VM, Akey IV, Schmidt-Zachmann MS, Head JF, Akey CW. (2004) Structure 12, 2149-2160.[3] Lee HH, Kim HS, Kang JY, Lee BI, Ha JY, Yoon HJ, Lim SO, Jung G, Suh SW. (2007) Proteins 69, 672-678.[4] Grummitt CG, Townsley FM, Johnson CM, Warren AJ, Bycroft M. (2008) J. Biol. Chem. 283, 23326-32.[5] Franco G, Bañuelos S, Falces J, Muga A, Urbaneja MA. (2008) Biochemistry 47, 7954-7962.[6] Taneva SG, Bañuelos S, Arregi I, Falces J, Konarev P, Svergun D, Velázquez-Campoy A, Urbaneja MA. (2009) J. Mol. Biol. 393, 448-463.

contactauthor:BenoitlecTez

187


poster 80

replacing arg149 by Cys in the melibiose permease hampers one step in the transport cycleYi-Bin Lin1,2, Oliver Fuerst1,2, Víctor A. Lórenz-Fonfría1,2, Meritxell Granell1,2, Gérard Leblanc3, and Esteve Padrós1,2

1Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medici-na.2Centre d’Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelo-na, Spain.3Laboratoire de Physiologie des Membranes Cellulaires-LRC-CEA 16V, Université de Nice Sophia-Antipolis and CNRS (UMR 6078), 06238, Villefranche sur Mer Cedex, France

The melibiose permease (MelB) from Escherichia coli is a sugar/cation co-transporter mem-brane protein that uses the electrochemical gradient of H+, Na+ or Li+ to drive the uptake of the disaccharide melibiose [1]. Previous sugar transport and binding assays in cells and RSO (Right-side-out) membrane vesicles suggested that replacing Arg149 in the cytoplasmic loop 4-5 of MelB by a neutral Cys residue leads to inactive transporter [2]. Here, we performed further experiments on MelB reconstituted into liposomes (Inside-side-out, ISO) [3], aiming to understand the role of Arg-149 in the transport mechanism of MelB. Fourier transform infrared (FTIR) difference spectroscopy [4, 5] and fluorescence spectroscopy [2, 3] have been combined to obtain information about substrate-induced structural changes of the MelB mu-tant R149C. Cooperative changes in MelB conformation upon substrate binding were inferred from the analysis of the intrinsic fluorescence of MelB and by using fluorescence resonance energy transfer spectroscopy (FRET) with a fluorescent sugar analog, Dns2-S-Gal. All the re-sults indicate that melibiose can bind to R149C, inducing fluorescence changes in ISO pro-teoliposomes but not in RSO vesicles [2]. These suggest that R149C can bind the externally added sugar analog in the ISO orientation, but not in the RSO orientation. Comparison of Na+-induced FTIR difference spectra of R149C and Cys-less show, as a main change, the disappea-rance of a positive band near 1671 cm-1 in R149C. This missing band could originate from the formation of a salt bridge by an Arg side chain upon Na+ binding, a process that could be missing in R149C. It is further shown that replacement of Arg149 by Cys decreases the intensity of peaks at 1663 and 1657 cm-1, corresponding to changes in α helices, indicating a decrease in α helical conformational changes or reorientations. Taken together, the results indicate that the mutation of Arg149 to Cys obstructs the protein conformational changes from an inward-facing orientation to the outward-facing orientation.

[1] Pourcher, T. et al. (1995) Biochem. 34, 4412-4420. [2] Manal-Abdel, D. et al. (2003) J Biol Chem. 280, 2721-2729.[3] Meyer-Lipp, K, et al. (2006) J Biol Chem. 281, 25882-25892.[4] Léon, X. et al. (2005) Biochem. 44, 3506-3514.[5] Léon, X. et al. (2009) Biophys J. 96, 4877-4886.

contactauthor:yi-BinlIn

188

poster 81

Cardiolipin, a key component to mimic the e. coli bacterial membrane in model systems membranesSílvia Lopes1, Cristina Neves1, Peter Eaton1, Paula Gameiro1

1Requimte, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal.

The phase transition temperatures of several lipidic systems were determined using two di-fferent techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize diffe-rent lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for E. coli membranes. Given the important role of cardiolipin (CL) in na-tural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared to those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.

contactauthor:sílvialopes

189


poster 82

Viscoelastic properties of natural ceramidesElisa R. Catapano1, Iván López-Montero1 and Francisco Monroy1

1Membrane mechanics and biorheology group. Departamento Quimica Fisica I. Universidad Complutense de Madrid

Ceramide levels on plasma membrane dramatically increase under cell stress activated by multiple agonist factors. Ceramides are then observed to be laterally seggregated into cerami-de rich domains and supposed to promote signalling protein capping. This putative role assig-ned to ceramides could be related to their mechanical properties. In this communication we describe the viscoelastic properties of egg ceramide monolayers studied by surface rheology. Compared to other common sphingolipids and phospholipids, ceramides show a very distinc-tive mechanical behaviour. Ceramides are characterised by a high compression modulus (200 mN/m), a very high shear modulus (90 mN/m) and a shear viscosity, which is several orders of magnitud higher than found for usual fluid lipid phases (including lipid mixtures in a liquid ordered phase). All this particular features emphasize the hard solid character of ceramide enriched domains and constitute them as excellent platforms for protein immobilisation.

contactauthor:Ivánlópez-MonTero

190

poster 83

Surfactant inhibition and reactivation as seen in captive bubble surfactometryElena Lopez-Rodriguez1, Olga L. Ospina1, Mercedes Echaide1, H.William Taeusch2 and Jesus Perez-Gil1

1Departamento de Bioquímica y Biología Molecular, Facultad de Biología, UCM2Department of Pediatrics, San Francisco General Hospital, UCSF

Pulmonary surfactant is a complex mixture of lipids and proteins located at the air-water interface in the alveoli. The main function of pulmonary surfactant is lowering the surface tension, thus decreases the work of breathing and prevents alveoli collapse. Dysfunction of surfactant in the lungs course with respiratory distress syndromes as for example ARDS (acu-te respiratory distress syndrome) or MAS (meconium aspiration syndrome). Many substances have been described as inhibitory agents for pulmonary surfactant, being the most common in vivo serum, cholesterol and meconium.

In Captive Bubble Surfactometry we can study biophysical relevant properties under physio-logical conditions. For instance we can follow the rapid film adsorption of the pulmonary sur-factant into the interface of a bubble. Moreover it is possible to monitorize the re-adsorption of the excessive material from the reservoir to the interface of an expanded bubble. And we can follow dynamic properties during compression-expansion cycling.

We have analyzed the effect of the main inhibitory agents (serum, meconium and cholesterol) in the different biophysical properties of lung surfactant as seen in CBS. Therefore we are able to observe differences in the presence or absence of these agents in interfacial adsorption, post-expansion adsorption and compression-expansion cycling. Moreover we have studied the effects of adding polymers to pulmonary surfactant in order to prevent inhibition as a new therapeutic option to be considered.

contactauthor:elenalopez-rodrIguez

191


poster 84

identification of some inhibitors compounds of β - amyloid aggregation.Laura Catalina López1, José Alberto Carrodeguas1, Salvador Ventura2, Javier Sancho1.1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Universidad de Zaragoza.2Departament de Bioquímica i Biologia Molecular, Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona.

Alzheimer’s disease is the most common cause of loss of cognitive function in older people. It is characterized by the presence of numerous senile plaques and neurofibrillary tangles and neuronal loss. The senile plaques are composed by β-amyloid peptide, which have between 39 and 43 residues [1, 2, 3].

Two mass screening of compound libraries, formed by a total of 11,200 compounds, were ca-rried out, which adhere to the Lipinski´s rules. For this purpose fluorescence spectroscopic techniques, light scattering, transmission electron microscopy and viability assays in HeLa cells were employed.

Several compounds that show an inhibitory effect on the aggregation of β-amyloid peptide were identified (17-40).

[1] Ray, I., Chauhan, A., Wegiel, J., and Chauhan, V.P.S. (2000) Brain Research 853, 344 - 351.[2] Szczepanik, A.M., Rampe, D., and Ringheim, G.E. (2001) Journal of Neurochemistry 77, 304 – 317.[3] Wiesehan, K., Stöhr, J., Nagel-Steger, L., van Groen, T., Riesner, D., and Willbold, D. (2008) Protein Engineering, Design & Selection. vol. 21, no 4, 241-246.

contactauthor:lauracatalinalópez

192

poster 85

Binding and stability of structural mimics of the phosphorylated species of the histidine phosphocarrier proteins of Streptomyces coelicolor.Miriam López-Pérez1, Ana-Isabel Martínez-Gómez2, David Aguado-Llera1, Sergio Martínez-Rodríguez2, Javier Gómez1 and José L. Neira1,3

1Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche.2Departamento de Química-Física, Bioquímica y Química Inorgánica, Universidad de Alme-ría, Almería.3Instituto de Biocomputación y Física de los sistemas complejos, Zaragoza.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS), only found in bacteria, is responsible for the detection, migration towards, and simultaneous phosphorylation and uptake of carbohydrates involved in the metabolism of the cell. The PTS catalyses the transfer of an activated phosphoryl group from phosphoenolpyruvate (PEP) to the imported carbo-hydrate, through a cascade of five proteins. This transference occurs starting from PEP, via phosphointermediates of the general phosphotransferases enzyme I (EI) and the histidine-containing phosphocarrier (HPr) to substrate-specific enzyme II permeases (the so-called IIA-BCsug). These proteins are also involved in regulation of sugar uptake at the level of gene expression or directly influence the catalytic activities of other proteins and antibiotic regu-lation. The EI protein is a dimer formed by 576-residues-long monomers, constituted by a C terminal and a N terminal fragment (EIC and EIN, respectively). We have cloned the isolated EIN fragment, which is 246-residues long.

In this work we first characterize the conformational and thermal stability of HPr mutants designed to mimic its phosphorilated species as well as the energetic of its molecular recog-nition by the phosphotransferase enzyme I (EI). In particular, the thermodynamic stability of the HPrH15D mutant has been studied by a variety of biophysical techniques, namely circular dichroism, steady-state fluorescence, and differential scanning calorimetry. Both its conformational and thermal stability are shown to be similar to that of wild-type. Finally, the energetics of the binding of this mutant to EIN has been characterized by isothermal titration calorimetry.

contactauthor:Miriamlópez-pérez

193


poster 86

Structural insights into the activation mechanism of melibiose permease by sodium bindingMeritxell Granell1, Xavier León1,3, Gérard Leblanc2, Esteve Padrós1 and Víctor A. Lórenz-Fonfría1.1Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, and Centre d’Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 2Institut de Biologie et Technologies-Saclay, Service de Bioenergétique, Biologie Structurale et Mécanismes, CEA-Saclay, F-91191 Gif sur Yvette, France

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of Na+ (or less effectively H+) [1, 2]. MelB is a biochemically well-characterized transporter [1], making it a convenient model to analyze the molecular and structural basis of the Na+ binding site and the Na+-coupled transport mechanism. An additional interest is that its yet unknown 3D structure is expected to follow the fold of the crystallized transporters of the MFS superfamily [3] rather than the one observed in all the Na+-symporters so far crystallized [4].

Functional studies using mutants established that four Asp residues located in the putative N-ter 6-helices bundle of MelB (Asp19, Asp55, Asp59, Asp124) are crucial for Na+ dependent affinity increase and transport of melibiose, leading to their assignment as Na+ ligands [2]. Nevertheless, direct evidence for this assignment is still missing.

In this work we reevaluate the role of these Asp residues in MelB, with special emphasis in their involvement in the Na+ and melibiose binding and in their coupling process. To do so we relayed on substrate-induced infrared difference (IRdiff) spectroscopy [5, 6]. In this technique, substrate-induced IRdiff spectra are measured in response to the interaction of the coupling ion with MelB (WT and mutants), or to the interaction of the sugar with the ion-MelB binary complex.

The results confirm firstly that Asp55 and Asp59 in helix II are essential ligands for Na+ binding. Secondly, Asp124 in helix IV is not essential for Na+ binding, but interestingly it is required for the full Na+-induced conformational changes presumably involved in the coupling mechanism. Asp124 appears also to be a sugar ligand. Thirdly, Asp19 in helix I does not participate in Na+ binding as previously thought, but it is a melibiose ligand. The location of these four residues in two indepen-dent constructed homology models of MelB is consistent with their above concluded role.

[1] T. Pourcher, M. Bassilana, H.K. Sarkar, H.R. Kaback and G. Leblanc, Philos. Trans. R. Soc. Lond. B: Biol. Sci. 326 (1990) 411-23.[2] B. Poolman, J. Knol, C. van der Does, P.J. Henderson, W.J. Liang, G. Leblanc, T. Pourcher and I. Mus-Veteau, Mol. Microbiol. 19 (1996) 911-22.[3] C.J. Law, P.C. Maloney and D.N. Wang, Annu. Rev. Microbiol. 62 (2008) 289-305.[4] H. Krishnamurthy, C.L. Piscitelli and E. Gouaux, Nature 459 (2009) 347-55.[5] X. León, V.A. Lórenz-Fonfría, R. Lemonnier, G. Leblanc and E. Padrós, Biochemistry 44 (2005) 3506-14.[6] V.A. Lórenz-Fonfría, M. Granell, X. León, G. Leblanc and E. Padrós, J. Am. Chem. Soc. 131 (2009) 15094-5.

contactauthor:Víctora.lórenz-FonFría

194

poster 87

the behavior of pyrene in popC and popC/cholesterol bilayers: a molecular dynamics studyLuís Loura1,2, António do Canto3,4

1Faculdade de Farmácia, Universidade de Coimbra, Portugal2Centro de Química de Coimbra, Universidade de Coimbra, Portugal3Departamento de Química, Universidade de Évora, Portugal4Centro de Química de Évora, Universidade de Évora, Portugal

Due to their sensitivity and versatility, the use of fluorescence techniques in membrane bio-physics is widespread. Because membrane lipids are non-fluorescent, extrinsic membrane probes are widely used. However, the behavior of these probes when inserted in the bilayer is often poorly understood, and it can be hard to distinguish between legitimate membra-ne properties and perturbation resulting from probe incorporation. Atomistic molecular dy-namics (MD) simulations present a convenient way to address these issues and have been increasingly used in recent years in this context [1]. In this work we use MD to simulate fully hydrated bilayers composed of 128 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 120 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine:30 cholesterol (POPC:Chol 4:1) molecules, with 0, 2 and 4 inserted pyrene molecules. Fluorophore properties such as mass distribution profile along the bilayer normal, orientation, extent of solvation, and rota-tional and translational dynamics are monitored, as well as bilayer parameters including area per lipid, mass distribution profiles, translational and rotational (headgroup, acyl chain) dyna-mics, order parameters, orientation of water molecules and lipid headgroups at the interface, and electrostatic potential across the membrane. In agreement with recent results reported for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) above the gel/fluid transition tem-perature [2], it is found that pyrene prefers to be located in the hydrophobic acyl chain region close to the glycerol group of lipid molecules and causes ordering of the lipid acyl chains. On the other hand, and similarly to one-component DPPC gel bilayers [2], incorporation of pyrene in binary POPC:Chol 4:1 bilayers produces a disordering effect, increasing the area/lipid molecule and decreasing the bilayer thickness and acyl chain order parameter, and thus opposing the ordering effect of cholesterol.

[1] Loura, L.M.S., and Prates Ramalho, J.P. (2009) Biophys. Rev. 1, 141-148. [2] Curdová, J, Capková, P, Plášek, J, Repáková, J, and Vattulainen, I (2007) J. Phys. Chem. B 111, 3640-3650

contactauthor:luísloura

195


poster 88

a molecular dynamics study of ligand imprintingDiana Lousa1; António M. Baptista1; Cláudio M. Soares1

1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157 Oeiras, Portugal

When an enzyme is placed in an anhydrous apolar solvent after being in contact with a inhi-bitor, it appears to retain the state induced by the ligand. This curious phenomenon is known as “ligand imprinting” and was first reported by Russell and Klibanov in 1988 [1]. Moreover, these authors found that in water the pre-treatment with the ligand has no effect.

In this work we analyze the molecular determinants of ligand imprinting using a molecular dynamics (MD) methodology. We started by docking an inhibitor in the active site of the serine protease subtilisin. In order to adapt the active site to the inhibitor, the enzyme-ligand complex was placed in water and 20 ns of MD simulations were carried out. The ligand was then removed and the resulting structure was subjected to 10 ns of MD simulations in hexane and in water. As a control, we performed MD simulations in the same solvents, but starting from a “ligand untreated” structure of subtilisin. Several replicates were used in all the condi-tions tested to avoid lack of sampling problems.

Our results show that pre-treating the enzyme with the inhibitor in hexane increases the probability of finding an open active site. On the other hand, in water the active site of the enzyme in the “pre-treated” simulations is indistinguishable from the control case. Analyzing the protein fluctuations, we could conclude that the observed behavior reflects the fact that subtilisin is considerably more rigid in hexane than in water and therefore, in the apolar sol-vent, it tends to get “locked” in the conformation induced by the ligand.

[1] Russell, A.J. and A.M. Klibanov (1988) J. Biol. Chem. 263, 11624-11626.

contactauthor:Diana LOUSA

196

poster 89

Membrane pore formation by the human immunodeficiency virus type-1 neutralizing anti-gp41 antibody 2f5Rubén Maeso‡, Renate Kunert§, and José L. Nieva‡

‡Biophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular Biology Department, niversity of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain.§Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria

A prominent feature of HIV-1 neutralizing monoclonal antibody (MAb) 2F5 is the presence of an unusually elongated and hydrophobic CDR H3 loop, which establishes not major contacts with bound epitope residues. Thus, it has been hypothesized that this loop might enable di-rect antibody association with the membrane surface, a step that would be required for the subsequent binding of membrane-inserted gp41 epitope sequences. In addition, due probably to the combination of the loop’s hydrophobicity with an overall positively charged paratope surface, this MAb has been shown to associate with anionic phospholipids, specifically car-diolipin (CL). In this contribution we describe a new Mab2F5 property, its capacity to directly porate membranes.

contactauthor:Rubén MAESO

197


poster 90

Constant-ph molecular dynamics simulation with explicit membranes: study of peptide-bilayer interactionsP. R. Magalhães1, M. Machuqueiro2, A.M. Baptista1

1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal 2Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal

Biological membranes are among the most important of structures, comprising the boun-daries that define the inside and outside of a cell. During the last decades, the availability of powerful computers has opened new ways of studying lipid bilayers in atomic detail, yielding detailed pictures of their structure and dynamics [1]. However, the accurate incorporation of important variables such as pH in membrane simulations remained a difficult challenge to overcome. One way to solve this problem is through the use of the so-called constant-pH molecular dynamics (MD) methods, therefore we altered the latest implementation [2] of our stochastic titration method for constant-pH MD [3] in order to allow the explicit inclusion of membranes in the simulations.

We present the first constant-pH MD study of an explicit membrane system, simulating kyo-torphin (KTP), an endogenous dipeptide [4], in the presence of a 128 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) lipid bilayer and aqueous solvent. pH values between 2 and 12 were studied using a constant-pH MD method. For each pH value, three replicated were run, each 100 ns long.

We followed the area and volume per lipid parameters in order to acess the stability of our membrane and found them to be in accordance with experimental values. We also found that KTP preferred the lipid phase over the solvent and measured the degree of insertion in the membrane, finding it to be independent of pH. The conformation distribution of the peptide in the membrane was also analyzed, and a pH dependence was observed, which is in accordance with recent observations in water [5]. Additionally, the pKa values for the four titratable sites of KTP were calculated and found to be similar to those previously obtained in water.

[1] Ash, W.L., Zlomslic, M.R. Oloo, E.O., Tieleman, D.P. (2004) Biochim. Biophys. Acta 1666: 158-189.[2] Machuqueiro, M and Baptista, A.M. (2008) Proteins: Struct. Funct. Bioinf. 72: 289-298.[3] Baptista, A.M., Teixeira, V.H. and Soares, C.M. (2002) J. Chem. Phys. 117, 9: 4184-4200.[4] Takagi, H. et al., (1979) Eu. J. Pharm. 55: 109-111.[5] Machuqueiro, M. and Baptista, A.M. (2007) Biophys. J. 92 : 1836-1845.

contactauthor:P. R. MAGALHãES

198

poster 91

Steady-state fluorescence quenching with pyrenyl probes in fluid bilayers examined by a two-dimensional kinetic formalismMiguel Manuel1, Jorge Martins1,2

1IBB-CBME (Institute for Biotechnology and Bioengineering - Center for Molecular and Struc-tural Biomedicine), University of Algarve, Faro, Portugal.2DCBB-FCT (Department of Biological Sciences and Bioengineering – Faculty of Sciences and Technology), University of Algarve, Faro, Portugal.

We studied the excimer formation of a phospholipid-labeled pyrenyl probe in pure POPC and POPS multilamellar vesicles, along with defined mixtures of both phospholipids, at 25ºC, 35ºC and 45ºC. Excimer formation in POPC bilayers is in good agreement with the theoretical predictions by a kinetic formalism specific for steady-state fluorescence quenching processes occurring in two-dimensional media [1]. Results in mixtures of POPC with 10% and 20% POPS were practically indistinguishable from those obtained with pure POPC vesicles, however excimer formation in pure POPS bilayers appears to be appreciably higher.

Paving on the known details about the physical chemical factors that determine the incorpo-ration of 1-pyrenesulfonate (PSA) into model membranes and location within the bilayer [2], we have also investigated the quenching of PSA by low concentrations of a DOXYL quencher group covalently linked to an acyl phospholipid chain (5-DOXYL-PC) in fluid POPC bilayers. The results were also scrutinised by the same two-dimensional kinetic formalism [1].

[1] Razi Naqvi, K., Martins, J., Melo, E. (2000) J. Phys. Chem. B, 104, 12035-12038.[2] Manuel, M., Martins, J. (2008) Chem. Phys. Lipids. 154, 79-86.

contactauthor:Miguel MANUEL

199


poster 92

Dependence of mean evolutionary time with the mutation rateArturo Marín1, Hector Tejero1, Juan Carlos Nuño2 and Francisco Montero1.1Department of Biochemistry and Molecular Biology I. Universidad Complutense de Madrid (UCM), 28040-Madrid, Spain.2Department of Mathematics Applied to Natural Resources, Escuela Técnica Superior de Ingenieros de Montes, Universidad Politécnica de Madrid, 28040-Madrid, Spain.

The time that an error-prone selfreplicative system take to go from one quasispecies to ano-ther with higher selective value must depend on the mutation rate [1]. In fact, some pre-vious results obtained from stochastic models, in which the evolution time is quantify by measuring the time when the target sequence shows up for the first time in the population, had demonstrated that optimal mutation rate exist [1]. The aim of this communication is to explore, from a quantitative point of view, the dependence of the optimal evolutionary time on the differents parameters of the system such as the shape and other properties of fitness-landscape. At this end, deterministic models in the framework of those proponed by Eigen [2] have been developed, and in all the cases an average evolutionary time have been evaluated as previously described [3]. The importance of the optimal mutation rate in the process of evolution and selection is discussed.

[1] Stich M, Briones C, and Manrubia S (2007). Collective properties of evolving molecular quasispecies. BMC Evolutionary Biology, 7:110, 1-13.[2] Eigen, M. (1971). Selforganization of matter and the evolution of biological macromolecules. Na-turwissenschaften. Volume 58, Number 10, 465-523.[3] Lloréns M, Nuño JC, Rodríguez Y, Meléndez-Hevia E, and Montero F (1999). Generalization of the Theory of Transition Times in Metabolic Pathways: A Geometrical Approach. Biophysical Journal, Volu-me 77, 23–36.

contactauthor:arturoMarín

200

poster 93

Molecular Dynamics analysis of the conformational change of paramyxovirus F protein in the initial steps of membrane fusion.Fernando Martín-García1,2, Jesús I. Mendieta-Moreno1 , Jesús Mendieta1,2 and Paulino Gómez-Puertas1. 1Molecular Modelling Group, Centro de Biología Molecular “Severo Ochoa” (CBMSO, CSIC-UAM). Cantoblanco, 28049 Madrid - Spain.2Biomol-Informatics SL. C/ Faraday, 7. Parque Científico de Madrid. Cantoblanco, 28049 Madrid - Spain.

The paramyxovirus family is composed of enveloped, negative-stranded RNA viruses, many of which are human pathogens (i.e. measles virus, human respiratory syncytial virus, Hendra and Nipah viruses or human metapneumovirus). Their entry into target cells is mediated by two glycoproteins located on the viral membrane: the attachment protein and the fusion (F) protein. The F protein belongs to the class I of integral membrane proteins and it is initially synthesized as F0 precursor subsequently cleavaged into the active F1+F2 heterodimer. After binding to the host cell membrane, trimeric F1+F2 complex experiments an irreversible and large structural change resulting in a completely different post-fusion structure when compa-red with the pre-fusion state. Crystal structures has been recently solved for both pre-fusion (Parainfluenza virus 5; PDB code: 2B9B) and post- fusion (human Parainfluenza virus 3; PDB code: 1ZTM) conformations illustrating this dramatic change. In contrast to the well known mechanism of fusion event of orthomyxovirus, no changes in pH are required in the case of paramyxovirus and, unfortunately, solved structures has not given a suitable clue for the me-chanism of the initial fusion triggering event.

Taking advantage of biased molecular dynamics techniques, using AMBER10 package, we have simulated the first steps of the F protein changes after binding to the host membrane. Starting from the published structure of pre-fusion state (PDB code: 2B9B), we have simulated the initial stretching of the HRA region after cleavage of the activation site. After complete elongation of the folded peptide (including a pair of beta strands and a short alpha helix structures), we have also simulated the posterior shortening of the extended structure. During the shortening process, the peptide showed a spontaneous rearrangement in a completely helical structure, comparable to the obtained by crystallization of the post-fusion state. In a subsequent step, a trimeric intermediate composed by three helical structures obtained as above, was subjected to un-biased molecular dynamics, resulting in the rapid formation of a triple coiled-coil helical structure, equivalent to the final, stable structure of the peptide in the post-fusion protein (PDB code: 1ZTM). As this complete recreation of the initial states of the fusion triggering events of paramyxovirus F protein has been obtained simulating the effect of only mechanical forces, without changes in temperature or pH, the results also allow us to hypothesize that the conformational change can be produced only by mechanical causes, i.e. the movement generated by the insertion of the small fusion peptide in the host membrane followed by short-term agitation of the virus close to the cell surface.

contactauthor:FernandoMarTín-garcía

201


poster 94

Spectroscopic Studies on the interaction of the cationic conjugated polyelectrolyte poly{[9,9-bis(6’-n,n,n-trimethylammonium) hexyl] fluorene-phenylene} bromide (htMa-pFp) with human Serum albumin.Maria José Martínez-Tomé, Rocío Esquembre, Ricardo Mallavia and C. Reyes Mateo. .

Instituto de Biología Molecular y Celular. Universidad Miguel Hernández de Elche03202 Elche (Alicante), Spain

Fluorescent conjugated polyelectrolytes constitute an interesting class of materials with a wide range of properties and potential applications in areas such as chemical and biological sensing. The present study reveals the strong and non-specific interactions that can exist bet-ween this class of polymers and human serum proteins and that must be taken into account in order to define the real-world sensing applications. In particular, the work uses different approaches to explore the interaction between the cationic conjugated polyelectrolyte HTMA-PFP and the protein HSA in aqueous solution. Such interaction has been investigated from changes observed in both, the spectroscopic properties of HTMA-PFP and the intrinsic fluo-rescence of HSA. Absorption and fluorescence spectra of HTMA-PFP suggest that, at low con-centration of protein, HTMA-PFP and HSA form a polymer-protein complex due to electros-tatic interactions between the cationic side chains of HTMA-PFP and the negatively charged surface of the protein (pI = 4.9). Interaction between both macromolecules is accompanied by an increasing of the fluorescence signal of HTMA-PFP, which suggests that hydrophobic inte-ractions between the conjugated polymer backbone and the hydrophobic patches of HSA also contribute to the polymer-protein complex stabilization. In addition, this interaction produces a decreasing in the intrinsic fluorescence of HSA which is partially due to static quenching and energy transfer mechanism between both macromolecules. The fact that domains I and II of the protein are more acidic that domain III suggests that the electrostatic interaction occurs mainly between the polymer and the domains I and II. This hypothesis is supported from the thermal behaviour of the polymer fluorescence which is highly sensitive to the different steps involving HSA unfolding as well as from the protein quenching experiments, given the loca-tion of Trp-214 in domain II. These results were complemented by CD experiments of the HSA and its polymer complexes which indicate that the molecular interaction between HTMA-PFP and HSA decreases the stability of the protein, producing a loss of helical secondary structure content. Such changes seem to induce an alteration in polymer conformation which is signa-lled by an enhancement in its fluorescence intensity.

Acknowledgements: The authors thank the Spanish Ministerio de Ciencia e Innovación (MICINN) for grant MAT2008-05670. R. Esquembre acknowledges the support of a predoctoral fellowship from MI-CINN.

contactauthor:MariajoséMarTínez-ToMé

202

poster 95

thermodynamics of chlorpromazine association with lipid bilayersPatrícia A. T. Martins1, Adrián Velazquez-Campoy2, Winchil L. C. Vaz1 and Maria J. Moreno1

1Biological Chemistry Group, Department of Chemistry – FCTUC, University of Coimbra, 3004-535 Coimbra, Portugal2Institute of Biocomputation and Physics of Complex Systems, University of Zaragoza, 50009 Zaragoza, Spain

Psychomimetic drugs must permeate the tight epithelia of the vascular endothelium that constitutes the Blood Brain Barrier (BBB) and this is one of the major determinants in the development of new drugs. In order to quantitatively describe this permeation, our group has been working for several years in characterizing the kinetics and thermodynamics of the interaction of small amphiphiles with membrane model systems (insertion/desorption and translocation) [1-4].

Chlorpromazine (CPZ) is an antipsychotic drug used in the treatment of schizophrenia and is an ideal candidate for studies of passive permeation of the BBB by active drugs.

In order to assess the partition and translocation of CPZ in lipid membranes, we have used Isothermal Titration Calorimetry (ITC) to study the interaction of this drug with lipid vesi-cles composed of pure POPC, POPC:POPS (9:1) and POPC:Chol:POPS (6:3:1) employing two different protocols: uptake and release of the drug into and from lipid vesicles (See [5]). We have obtained the partition coefficients (KP) for CPZ into lipid vesicle and the enthalpy change (ΔH) associated with this process and gained some insight on the translocation rate, namely if it is faster or slower than the characteristic time of the ITC experiment.

The enthalpy change upon partition to all lipid bilayers studied is negative. Due to the positive charge of CPZ, the presence of negatively charged lipids in the bilayer increases both KP and the magnitude of ΔH. The presence of cholesterol decreases strongly the affinity of CPZ for the bilayer.

From the global fit of the experiments with the uptake and release protocols we conclude that the translocation in POPC at 37 ºC occurs within a few minutes, while for all other systems, at 37 ºC and 25 ºC, it is negligible during the time required for baseline recovery in each injection of the ITC experiments (a few minutes).

[1 ]Abreu, M. S. C., Moreno, M. J. and Vaz, W. L. C. (2004) Biophys. J. 87, 353-365[2] Sampaio, J. L., Moreno, M. J. and Vaz, W. L. C. (2005) Biophys. J. 88, 4064-4071[3] Moreno, M. J., Estronca, L. M. B. B. and Vaz, W. L. C. C. (2006) Biophys. J. 91, 873-881[4] Estronca, L.M. B. B., Moreno, M. J. and Vaz, W. L. C. (2007) Biophys. J. 93, 4244-4253[5] Tsamaloukas, A. D., Keller, S. and Heerklotz, H. (2007) Nat. Prot. 2, 695-704

contactauthor:Patrícia A. T. MARTINS

203


poster 96

Structural basis of the FMn midpoint reduction potential modulation in anabaena FlavodoxinMarta Martínez-Júlvez1,2, Beatriz Herguedas1,2 and Milagros Medina1,2. 1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universi-dad de Zaragoza, 50009 Zaragoza, Spain.2Institute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, 50009 Zaragoza, Spain.

The crystallographic structures of some mutated variants of flavodoxin (Fld) from Anabaena in positions 59 and 92 have been determined in the oxidised state. Fld substitutes for ferre-doxin (Fd) in donating electrons from PSI to ferredoxin-NADP+/H reductase (FNR) when the cyanobacterium is grown under low iron conditions. Ile59 and Ile92 are the only two hydro-phobic residues exposed to solvent in the isoalloxazine ring environment where interaction of Fld with its ET partners is expected. Single mutational studies at these positions indicated that the nature of these side chains tunes the flavin midpoint potentials, therefore modulating the ET between photosystem I (PSI) and Fld [1]. When Ile59 and Ile92 were simultaneously replaced with Ala or Glu impaired FMN binding to the apoprotein was observed, while Esq/hq shifted to less negative values and Eox/sq to more negative ones [2]. Here we discuss these observations based on the analysis of the crystallographic structures of I59E, I59A/I92A and I59E/I92E Flds, particularly in the slight differences in the strength of the H-bond interactions between the N(5) of the flavin and the Asn58-Ile59 bond observed among the mutants and wild type protein. Such differences, together with other minor structural details, may explain the observed distortion of the FMN midpoint reduction potentials with regard to the wild type protein. These studies additionally contribute in the understanding of the factor that determinate the isoalloxazine midpoint reduction potentials within proteins.

[1] I. Nogues, M. Martínez-Júlvez, J.A. Navarro, M. Hervás, L. Armenteros, M. A. de la Rosa, T.B. Brodie, J. K. Hurley, G. Tollin, C. Gómez-Moreno, M. Medina, (2003) Biochemistry 42, 2036–2045.[2] S. Frago, G. Goñi, B. Herguedas, J. R. Peregrina, A. Serrano, I. Pérez-Dorado, R.l Molina, C. Gómez-Moreno, J. A. Hermoso, M. Martínez-Júlvez, S. G. Mayhew and M. Medina (2007) Arch. Biochem. Biophys. 467, 206-217.

contactauthor:Marta MARTíNEZ-JúLVEZ

204

poster 97

internalization and releasing of cholesterol in the endosome: stability and importance of the fourth and fifth binding module of the LDL receptor.Juan E. Martínez1,2, Xabier Arias-Moreno1,2, Adrián Velazquez-Campoy1 and Javier Sancho1,2

1Biocomputation and Complex Systems Physics Institute (BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain.2Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universi-dad de Zaragoza,50009 Zaragoza, Spain.

La internalización de lipoproteínas de baja densidad mediada por su receptor( r-LDL) es el principal mecanismo por el que las células incorporan colesterol. Se ha demostrado, que los módulos cuarto y quinto del dominio de unión del receptor (LR4 y LR5) resultan especialmen-te claves en la unión con las lipoproteínas [1].

Estudios recientes de nuestro grupo de investigación [2,3] y que han sido corroborados por otros grupos [4], pusieron de manifiesto el papel crucial que desempeña el calcio en la es-tabilidad del LR5. Este hecho unido a la baja estabilidad de LR5 a pH 5.5 sugieren que el desplegamiento del LR5 en el endosoma tardío podría mediar en el mecanismo de liberación de LDL. Así, la inestabilidad de los módulos del dominio de unión del r-LDL, en el endosoma, provocaría el desplegamiento de los mismos y la liberación de las LDL. Posteriormente, este dominio de unión autointeraccionaría con el dominio homólogo a EGFP del propio receptor, favoreciendo el reciclaje del mismo a la membrana.

Además, estudios aún en marcha están arrojando datos mediante espectroscopía y calorime-tría [5] sobre estabilidad y unión a calcio para el módulo cuarto del dominio de unión (LR4) y un constructo de los módulos 4 y 5 (LR4-5) que apuntan también a la importancia del calcio en el mantenimiento de la estructura funcional de ambos módulos.

Por otro lado, estudios preliminares sobre la cinética de liberación de calcio, están mostrando que se trata de un proceso rápido, coherentemente con el mecanismo propuesto.

[1] Fisher C, Abdul-Aziz D, Blacklow SC.Biochemistry. 2004 Feb 3;43(4):1037-44[2] Arias-Moreno X, Cuesta-Lopez S, Millet O, Sancho J, Velazquez-Campoy A. Proteins. 2010 Mar;78(4):950-61.[3] Arias-Moreno X, Velazquez-Campoy A, Rodríguez JC, Pocoví M, Sancho J. J Biol Chem. 2008 Aug 15;283(33):22670-9. Epub 2008 Jun 23[4] Zhao Z, Michaely P.Biochemistry. 2009 Aug 4;48(30):7313-24.[5] Velázquez-Campoy A, Ohtaka H, Nezami A, Muzammil S, Freire E. Curr Protoc Cell Biol. 2004 Sep;Chapter 17:Unit 17.8. Review.

contactauthor:Juan E. MARTíNEZ

205


poster 98

Molecular recognition of azathilones by microtubules.Ruth Matesanz1, Fabian Feyen2, Karl-Heinz Altmann3 and J. Fernando Díaz1.1Chemical and Physical Biology Department. C.I.B. (C.S.I.C.) Madrid (Spain)2Institute of Biochemistry and Molecular Medicine. University of Bern. (Switzerland)3Department of Chemistry and Applied Biosciences. Institute of Pharmaceutical Sciences. (ETH) Zürich. (Switzerland)

Azathilones are non-natural natural products related to epothilones [1] which are potent micro-tubule-stabilizing agents (MSA). Compounds from the epothilones family are in clinical trials as candidates for anticancer chemotherapy and one compound has already reached the market. Epo-thilones are described to bind to the paclitaxel luminal site in the microtubules (hidden from the solvent) [2] but little is known about the structural pathway they use to reach this site.

A fluorescent derivative of azathilone NBD(nitro-benzoxadiazole)-azathilone retains the MSA activi-ty of the parent compounds: it binds to microtubules, promotes tubulin assembly, inhibits cell proliferation in vitro and arrests the cell cycle at G2/M [3]. On top of that binding of this NBD-azathilone to microtubules raises both fluorescence emission and fluorescent anisotropy values [3], which make it a useful tool to study the binding kinetics of epothilones to microtubules.

We have analyzed the binding of NBD-azathilone to mi-crotubules in order to obtain information on the way it reaches the luminal non-exposed site. Binding is very fast and thus not compatible with the direct binding to the paclitaxel luminal site, as has been described for taxanes [4], indicating that epothilones might also bind in a two-step mechanism through an external, easily-accesible site.

Analysis of the kinetic data indicates that binding of the probe proceeds in two phases, binding to an ex-posed site followed by a translocation to the final site as described for taxanes [5]. Detailed analysis of the kinetic behaviour of the reaction indicates a complex

kinetic pathway, suggesting two possible different binding modes; these findings are in good con-cordance with other ongoing NMR studies on the binding mode of epothilones and azathilones.

[1] Feyen, F., Gertsch, J., Wartmann, M. and Altmann, K.H. (2006) Angew. Chem. Int. Ed. Engl. 45, 5880-5885.[2] Forli, S., Manetti, F., Altmann, K.H. and Botta, M. (2009) ChemBioChem. 4, 1-6[3] Gertsch, J., Feyen, F., Bützberger, A., Gerber, B., Pfeiffer, B. and Altmann, K.H. (2009) ChemBioChem. 10, 2513-2521[4] Diaz, J.F., Strobe. R., Engelborghs, Y., Souto, A.A. and Andreu, J.M. (2000) J. Biol. Chem. 275, 26265-26276 [5] Barasoain, I., García-Carril, A.M., Matesanz, R., Maccari, G., Trigili, C., Mori, M., Shi, J.Z., Fang, W.S., Andreu, J.M., Botta, M. and Diaz, J.F. (2010) Chem. Biol. 17, 243-253

contactauthor:Ruth MATESANZ

206

poster 99

the role of gln 61 in ras p21 gtp hydrolysis: a QM/MM studyFernando Martin-Garcia1,2, Paulino Gomez-Puertas1 and Jesus Mendieta 1,2

1Molecular Modelling Group, Centro de Biología Molecular “Severo Ochoa” (CBMSO, CSIC-UAM). Cantoblanco, 28049 Madrid - Spain.2Biomol-Informatics SL. C/ Faraday, 7. Parque Científico de Madrid. Cantoblanco, 28049 Madrid - Spain.

Ras p21 protein is one of the better characterized members of the G proteins. The proteins of this family are involved in cellular signal transduction [1], acting as conformational switches. Although it is known that the functionality of G proteins depends on its GTPase activity, the precise catalytic mechanism of GTP hydrolysis is still unclear [2]. A controversial point is the nature of the residue acting as the general base in the activation of the catalytic water. We use the hybrid QM/MM potential [3] implemented in AMBER10 package in order to get insight about the water activation mechanism inside of the protein moiety. In this work, we develop an approach based on the adaptively biased MD (ABMD) [4] in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. This approach pre-sents some characteristics of steered MD and also of umbrella sampling procedures, allowing the testing of several hypothesis of the catalytic mechanism of GTP hydrolysis. Our results show that the most favorable mechanism involves both Gln61 residue and two water molecu-les, being GTP the final proton acceptor, thus supporting the hypothesis of substrate-assisted catalysis [5].

References:[1] Neves, S.R., Ram, P.T. & Iyengar R. (2002) Science 296,1636-1639 [2] Wittinghofer, A. (2006) Trends Biochem. Sci. 31,20-23[3] Babin, V.,Roland, C. & Sagui, C. (2008) J. Chem. Phys. 128, 134101-134107[4] Kamerlin, S.C., Haranczyk, M. & Warshel, A. (2009) J. Phys. Chem. B. 113, 1253-1272[5] Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer, H.R. & Wittinghofer, A. (1995) Nature Struct. Biol. 2, 36-44

contactauthor:Jesus MENDIETA

207


poster 100

nanostructuration of Ferritin nanoparticles on Sensors using Dip-pen nanolithographyRocío de Miguel1, Elena Bellido2, María José Martínez-Pérez3, Daniel Maspoch2, Javier Sesé1, Carlos Gómez-Moreno1, Fernando Luis3, Daniel Ruiz-Molina2, Anabel Lostao1

1Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain.2Centro de Investigación en Nanociencia y Nanotecnología, CSIC-ICN, UAB, Spain.3Instituto de Ciencia de Materiales de Aragón, CSIC, Zaragoza, Spain.

Ferritin (Fn) is a spherical protein nearly ubiquitous in biology where it functions to direct the biomineralization of iron as a mechanism for maintaining iron homeostasis. Mammalian Fn is comprised of two classes of subunits, H-chain, that catalyzes the oxidation of Fe2+ to Fe3+ with molecular O2, and L-chain that comprises the mineral nucleation site; both self assemble to form hetero-24mers of 12 nm. The cage-like architecture of Fn provides an ideal size-constrained reaction environment for nanomaterial synthesis with the protein shell acting both to direct mineralization and as a passivating layer, preventing unwanted particle–particle interactions.

Our group has synthesized magnetic iron oxides and antiferromagnetic cobalt oxides nano-particles in apoFn controlling size and shape. These materials have been studied using low temperature physical techniques. As an illustration, we report experimental results showing magnetic measurements performed over two-dimensional arrays of antiferromagnetic 2 na-nometer sized CoO filled Fn fabricated with Dip Pen Nanolithography (DPN) using a high sensitivity SQUID-microsusceptometer [1]. DPN is a direct-write scanning probe lithography in which the AFM tip is used to deliver molecules on surfaces with nanoscopic control and has become a powerful and versatile deposition technique for structuring a wide range of molecu-les on surfaces. Following this approach, a relatively accurate number of Fn entities have been successfully integrated onto pre-selected areas of the sensor. To explore the detection limits of this technique, with the ultimate goal to measure only a few nanoparticles, that may allow fundamental physical studies or the observation of new phenomenologies so far not observed for bulk materials, control over the spatial resolution and control on the number of deposited entities must be achieved. We are able to control the number of Fn molecules located on each spot [2], resulting in the control of the deposition of the containing nanoparticles [3]. Thanks to its biocompatibility and the capacity to bind molecules to its protein structure, Fn mate-rials allows us to deliver the cages to a targeted tissue or to achieve controlled assembly on a solid substrate to fabricate higher order structures. This makes Fn based nanoparticles ideal candidates for a broad range of materials with applications from biomedicine to electronics.

[1] Martínez-Pérez M. J., Sesé J., Luis F., Drung D, Schurig T. (2010). Review of Scientific Instruments, 81, 016108. [2] Bellido, E., R. de Miguel, R, Ruiz-Molina, D., Lostao, A., and Maspoch, D. (2010). Advanced Materials, 22 (3), 352-355.[3] Bellido, E., R. de Miguel, Sesé, J., R, Ruiz-Molina, D., Lostao, A., and Maspoch, D. (2010). Scanning, 32 (Issue 1), 35-41.

contactauthor:Rocío de MIGUEL

208

poster 101

reconstitution of bacterial cell division protein complexes in membrane-coated micro-beads: fluorescence spectroscopy studies. Begoña Monterroso1, Ariadna Martos1, Rubén Ahijado1, Carlos Alfonso1, Mercedes Jiménez1, Germán Rivas1, Silvia Zorrilla2 1Centro de Investigaciones Biológicas (CIB), CSIC. Ramiro de Maeztu, 9. 28040 Madrid, Spain.2Instituto de Química Física Rocasolano (IQFR), CSIC. Serrano, 117. 28006 Madrid, Spain.

Membrane coated micro-beads have been used to characterize the interactions between pro-teins involved in bacterial cell division by reconstructing them on the surface of the beads using either cytoplasmic membranes or polar extract lipids (that is, same lipid composition lacking protein content) from E. coli. These membrane-coated beads retain the capability of interaction with FtsZ, as monitored by fluorescence microscopy and other complementary tools. The GTPase FtsZ is the bacterial ancestor of tubulin and its GTP dependent assembly/disassembly cycle is essential for the formation of the septal ring in vivo [1,2]. We have also studied the interaction with the micro-beads of FtsA, an amphitropic protein widely con-served throughout bacteria and polydisperse in solution, involved in the E. coli proto-ring formation in the cytoplasmic membrane at the initial steps of the division ring assembly [1,2]. Co-sedimentation assays monitored by fluorescence measurements of the protein content after micro-beads pelleting showed important differences regarding binding affinity when using lipid or inner membrane as the coating material of the micro-beads. The interaction of FtsA with the natural inner membrane is stronger than with the E. coli lipids, suggesting a more favorable interaction with the lipids within the natural membrane environment and/or a net contribution from the native membrane proteins. The acknowledge of the participation of boundary conditions linked to diverse molecules as factors determining cellular behavior explains the growing importance of the cellular reconstitution of biological processes and quantitative synthetic biology fields [3].

[1] Vicente, M., Rico, A. I., Martínez-Arteaga, R., and Mingorance, J. (2006) J. Bacteriol. 188, 19-27.[2] Adams, D. W., and Errington, J. (2009) Nature Rev. Microbiol. 7, 642-653. [3] Liu, A. P., and Fletcher, D. A. (2009) Nature Rev. Mol. Cell Biol. 10, 644-650.

contactauthor:Begoña MONTERROSO

209


poster 102

partition of amphiphilic molecules to lipid bilayers by itC Maria João Moreno1, Margarida Bastos2 and Adrian Velazquez-Campoy3

1Chemistry Department FCTUC, 3004-535 Coimbra, Portugal. 2Chemistry Department, Faculty of Sciences (UP), P-4169-007 Porto, Portugal 3BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain, and Fundación ARAID, Diputación General de Aragón, Spain.

The partition of the amphiphile Sodium Dodecyl Sulfate between an aqueous solution and a POPC bilayer was followed by ITC as a function of the total concentration of SDS. It was found that the obtained partition coefficient is strongly affected by the ligand concentration, even after correction for the charge imposed in the bilayer by the bound SDS. The partition coeffi-cient decreased as the total concentration of SDS increased, this effect being significant for local concentrations of SDS in the lipid bilayer above 5 molar %. At those high local concen-trations, the properties of the lipid bilayer are strongly affected, leading to non-ideal behavior and concentration dependent apparent partition coefficients.

It is shown that with the modern ITC instruments available, the concentrations of SDS can be drastically reduced while maintaining a good signal-to-noise ratio. The intrinsic parameters of the interaction with unperturbed membranes can be obtained from the asymptotic behavior of the apparent parameters as a function of the ligand concentration, both for non-ionic and ionic solutes. A detailed analysis is performed, and an excel spreadsheet was developed to obtain the interaction parameters with and without correction for electrostatics.

contactauthor:Maria João MORENO

210

poster 103

the transcriptional regulatory network of Mycobacterium tuberculosisJoaquín Sanz1, Jorge Navarro2, Ainhoa Arbués3, Carlos Martín3, Pedro Marijuán2, Yamir More-no1,4.1Institute for Biocomputation and Physics of complex systems BIFI.2Instituto aragonés de ciencias de la salud IACS.3Grupo de genetica de mycobacterias, Facultad de Medicina, Universidad de Zaragoza.4Departamento de Física teórica, Universidad de Zaragoza.

The characterization of transcriptional regulatory (TR) networks beyond the context of model organisms offers, under the perspective of network science, the possibilities of a versatile tool which potential remains yet mainly unexplored. In this work, we present a new version for the TR network of Mycobacterium tuberculosis, which incorporates newly characterized transcriptional regulations coming from more than 40 recent, different experimental works available in literature. As a result of the incorporation of these new data -only feasible thanks to the proliferation of microarray experiments in the last years- our network doubles the size of previous data collections; incorporating more than a third of the entire genome of the microorganism. The assembling and rationalization of the experimental data that constitute our network, considering its volume and completeness, can constitute an important resource in diverse tasks like dynamic modeling, computational reliability determination or protein function prediction; being this point of particular relevance for the case under study, given that the function of only an small percent of the proteins of M.tuberculosis is known. Beyond the above mentioned, we present in this work an exhaustive topological analysis of the new assembled network, specially focusing on the search of topological, evolutionary trackers that will help us to improve our understanding of the interplay between topology and evolution in a strictly biological context.

contactauthor:Yamir MORENO

211


poster 104

Kinetic Modeling of Single Molecule Data: the phi29 Dna polymerase unwinding activity.José A. Morín1, 2, Borja Ibarra1, 2, J. Ricardo Arias-González1, 2, Margarita Salas3, José M. Val-puesta2, José L. Carrascosa2.1Imdea Nanociencia. Canto Blanco, Madrid 28049, Spain.2Centro Nacional de Biotecnología (C.S.I.C.), Canto Blanco, Madrid 28049, Spain.3Centro de Biología Molecular, ‘Severo Ochoa’ (C.S.I.C.-U.A.M.), Canto Blanco, Madrid 28049, Spain.

Mechanical force at the molecular level is involved in the action of many enzymes. During DNA replication the mechanical unwinding of the double-stranded DNA (dsDNA) helix is re-quired to separate the two complementary strands which are used by the DNA polymerases to replicate the DNA. In the bacteriophage Phi29 these two processes (replication and unwin-ding) are tightly coupled within the same protein. The Phi29 DNA polymerase unwinds the dsDNA as it replicates in a processive manner one of the strands, working as a polymerase and as a helicase at the same time.

Using Optical Tweezers [1] we developed a single-molecule mechanical assay to elucidate the physical mechanism by which this polymerase opens the DNA helix and the basis for the tight coupling between replication and unwinding. With this experimental setup we studied the response of the polymerase unwinding activity to increasing forces on the replication fork and, by the rational design of a replication hairpin, to the effect of high GC density sequence barriers.

In order to explain the observed force and sequence dependence of the unwinding reaction we adapted a kinetic model based on a random walker in continuous time along a discrete, periodic one-dimensional lattice [2, 3] and combine it with the formalism needed to include the effect of force on the kinetics of a single molecule reaction [4]. Our modeling indicates that the DNA unwinding activity of the Phi29 DNA polymerase is far from a purely passive motor, which should depend on the spontaneous thermal breathing of the DNA to advance. Instead, the data is consistent with an active mechanism in which the polymerase applies a destabilization energy that moves the double DNA helix unwinding reaction toward opening.

[1] Smith, S.B., Cui, Y. and Bustamante, C. (2003) Methods Enzymol. 361, 134–162.[2] Kolomeisky, A. B. and Widom, B. (1998) J. Stat. Phys. 93, N. 3-4. [3] Chemla, Y. R., Moffit, J. R. and Bustamante, C. (2008) J. Phys. Chem. B. 112, 6025-6044.[4] Tinoco Jr, I. and Bustamante, C. (2002) Biophysical Chemistry. 101 –102, 513–533.

contactauthor:José A. MORíN

212

poster 105

energetics of nucleotide-induced DnaK conformational states.Fernando Moro1, Stefka G. Taneva1, Adrián Velázquez-Campoy2 and Arturo Muga2

1Unidad de Biofísica (CSIC/UPV-EHU) y Departamento de Bioquímica y Biología Molecular. Facultad de Ciencia y Tecnología, Universidad del País Vasco, Apartado 644, 48080 Bilbao, Spain.2Instituto de Biocomputación y Física de Sistemas Complejos, Universidad de Zaragoza, Corona de Aragón 42, 50009 Zaragoza, Spain, and Fundación ARAID, Diputación General de Aragón, Zaragoza, Spain.

Hsp70 chaperones are molecular switches that use the free energy of ATP binding and hydro-lysis to modulate their affinity for protein substrates and, most likely, to remodel non-native interactions allowing proper substrate folding. By means of isothermal titration calorimetry, we have measured the thermodynamics of ATP and ADP binding to i) wild-type DnaK, the main bacterial Hsp70; ii) two single point mutants, DnaKT199A, which lacks ATPase activity but maintains conformational changes similar to those observed in the wild-type protein, and DnaKR151A, defective in interdomain communication; and iii) two deletion mutants, the isolated nucleotide binding domain (K-NBD) and a ΔLid construct (DnaK(1-507)). At 25ºC, ATP binding to DnaK results in a fast endothermic and a slow exothermic process due to ATP hydrolysis. We demonstrate that the endothermic event is due to the allosteric coupling bet-ween ATP binding to the nucleotide binding domain and the conformational rearrangement of the substrate binding domain. The interpretation of our data is compatible with domain docking upon ATP binding and shows that this conformational change carries an energy penalty of ca. 1 kcal mol-1. The conformational energy stored in the ATP-bound DnaK state, together with the free energy of ATP hydrolysis, can be used in remodeling bound substrates.

contactauthor:Fernando MORO

213


poster 106

Snapin is an oligomeric natively-unfolded protein, which forms a “fuzzy”, non-structured complex with snap-25 Aaron Navarro1, José A. Encinar1, Jesús Prieto2, Javie Gómez1, Alfonso Martínez-Cruz3, Grego-rio Fernández-Ballester1, José L. Neira1,4 and Antonio Ferrer-Montiel1 1Instituto de Biología Molecular y Celular; Universidad Miguel Hernández, 03202 Elche (Ali-cante); 2Departamento de Biología Estructural y Biocomputación, CNIO, 28029 Madrid3Structural Biology Unit, CIC bioGUNE, 48160 Derio (Vizcaya); 4Complex Systems Physics Institute, 50009 Zaragoza, Spain

Docking and fusion of synaptic vesicles are mediated by the assembly of a stable SNARE pro-tein complex. Snapin, a 15-kDa protein present in neuronal and non-neuronal cells, is a PKA-modulated protein that has been implicated in the regulation of the efficiency and kinetics of neurosecretion. Despite its pivotal role in cellular exocytosis, details of its structure, stability and conformational properties are limited. Here, we have addressed this issue and performed a structural and conformational characterization of wild type Snapin and the dimerization-defective, non-functional Cys66 to Ser (C66S) mutant, and investigated their binding to SNAP-25. Our results show that recombinant Snapin solutions are primarily composed of unfolded tetramers and dimers, with a low, non-stable, flickering α-helical structure, that irreversibly unfold at modest temperatures. In contrast, the C66S mutant appears as tetramers in equi-librium with trimers, with a higher content of α-helical secondary structure and increased thermal stability. Nonetheless, akin to wild type protein, the C66S mutant did not exhibit significant tertiary structure. Notably, we found that Snapin, but not the C66S mutant stably associated in vitro to recombinant SNAP-25 with an apparent affinity of 20 ± 10 μM and a 1:1 stoichiometry. However, neither SNAP-25 nor Snapin acquired a well-folded tertiary struc-ture upon binding, but they rather formed a “fuzzy” complex, as suggested by 1D-1H-NMR. Taken together, our findings support the notion that Snapin is a natively-unfolded oligomeric protein, and that this lack of, but inducible, protein structure appears to be important for its binding to the SNARE complex and other cellular partners. Furthermore, these results subs-tantiate the tenet that disulphide-mediated dimerization of Snapin monomers is essential for its interaction with the SNARE proteins.

contactauthor:aaronnaVarro

214

poster 107

Quantification of Synaptic Vesicles at the neuromuscular Junctions in Spinal Muscular atrophy MiceMargret Neher, Rocío Ruiz and Lucía Tabares

Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, 41009, España.

The motor neuron disease Spinal Muscular Atrophy (SMA) is the leading inherited cause of infant mortality. SMA is caused by mutation of the survival motor neuron1 (SMN1) gene and the resulting deficiency of the SMN protein. Recent studies suggest that the earliest detectable pathology in mouse models is at the neuromuscular junction synapse (NMJ). Surprisingly, most SMA NMJs remain innervated even late in the disease course but show abnormal synap-tic transmission.

We investigated the amount of synaptic vesicles in a mouse model of severe SMA (SMN∆7) in the rostral and caudal portions of the Levator Auris Longus muscle, at seven days of age. Through immunostaining we could visualize the vesicles using an antibody against the vesicu-lar acetylcholine transporter localized at the vesicle membrane together with a fluorescent se-condary antibody (Alexa 488). For the postsynaptic side we used bungarotoxine-rhodamine, a fluorescence dye against the acetycholine receptor.

The area of the post- and presynaptic site in SMA mouse were significantly smaller than in control littermates, but there was no detectable difference between the ratio of pre- and postsynaptic area in SMA and control littermates. This indicates that, at this age, NMJs in SMA mutant mice are smaller but the occupation by synaptic vesicles is normal.

contactauthor:Margret NEHER

215


poster 108

the nK1 receptor: Strategies of expression, purification and refolding.Mikhail Orel, Esteve Padrós, Joan Manyosa

Unitat de Biofísica, Dept. de Bioquímica i de Biologia Molecular i Centre d’Estudis en Biofisica, Universitat Autònoma de Barcelona, Spain

Neurokinin-1 is a membrane receptor that belongs to the family of GPCRs that has seven trans-membrane helices in its structure. The activation of this receptor by its ligands (Substance P, NKA, NKB) is involved in many biological processes, from activation of the immune system or the pain transmission to muscle contraction and vasodilation.

NK1R structure as well as the mechanism of interaction with its ligands is not well understood until now as a result of complexity of its obtention and purification in quantities sufficient for realizing studies by spectroscopic techniques. One of the possible strategies to obtain active NK1R in quan-tities required for the implementation of structural and functional studies is to use E.coli as an expression system. However, several attempts that have been made until now have shown that the NK1R is expressed in E.coli as inclusion bodies that can only be solubilized with strong ionic de-tergents such as SDS, or denaturing agents such as Guanidinium chloride (Gnd•Cl) resulting in the loss of the functions of the receptor.[1] The main objective of this study is to find the conditions for refolding of hNK1R expressed in E.coli, gradually decreasing the concentration of the denaturant agent. The refolding process of the receptor can be monitored by changes in the intrinsic fluores-cence of the tryptophan residues of the protein.

The E.coli strain C41 (DE3) was transformed with pET23b-hNK1R and receptor expression was induced by adding 1mM IPTG. After 3 hours of incubation, the culture was centrifuged and inclu-sion bodies were isolated by several centrifugation and washings. The inclusion bodies that were obtained were solubilized with 6M Gnd•Cl and then purified with column of His-select Nickel. The hNK1R functional form that has used as a reference in the refolding experiments was expressed in COS-1 cells, solubilized from membranes in 1% DDM and then purified with column of His-Select Nickel. [2]

The refolding of hNK1R was done in 50 mM TrisHCI buffer, pH 8.0, 100 mM NaCl with diffe-rent concentrations of Gnd•Cl (6M-0M) and SDS (1%-0%) to monitor the tryptophan fluorescence spectroscopy (λex 295 nm / λem 310-400 nm). The changes in turbidity of the samples were monitored at 400 nm by spectroscopy of UV-vis. The results show that in low concentrations of Gnd•Cl there is a blue shift in the emission spectrum peak that indicates changes of tryptophan to more hydrophobic environment as a result of protein folding. The shift becomes more evident if 0.1% DDM is added to the refolding buffer, indicating that the micellar structure of this non-ionic detergent stabilize the refolded receptor structure. In the experiments in which SDS was used, the emission spectra of the receptor presented weaker peak shifts (between 333nm and 331nm) which means that the NK1R is not fully denaturated in SDS and preserve part of its helical structure.

[1] Christova, P., Todorova K., Timcheva I., Nacheva G., Karshikoff A., and Nikolov P. (2003) Z. Natur-forsch 58c, 288-294. [2] Steven E. Bane, Javier E. Velasquet, Anne Skaja Robinson (2007) Protein expression and Purification 52, 348-355.

contactauthor:Mikhail OREL

216

poster 109

Modelling Spontaneous activity in neuronal CulturesJavier G. Orlandi1, Enric Álvarez-Lacalle2, Núria Amigó1, Jordi Soriano1 and Jaume Casade-munt1.1Departament d’Estructura i Constituents de la Matèria, Universitat de Barcelona, Av. Diago-nal 647, E-08028 Barcelona, Spain2Departament de Física Aplicada, Universitat Politècnica de Catalunya, Av. Dr. Gregorio Mara-ñón 50, E-08028 Barcelona, Spain

In the last decades neuronal cultures have emerged as a powerful tool to study and model collective phenomena in neuronal systems [1]. Cultures are typically prepared by extracting neurons from a rat embryonic brain and growing them ‘in vitro’. Although these neurons un-dergo a developmental pathway different than their ‘in vivo’ counterparts, they still preserve a large functionality. Cultured neurons start to grow and form connections with neighbouring cells within hours, and show spontaneous electrical activity (SA) already few days after pla-ting. At first, the SA is completely uncorrelated between neurons, i.e. they fire independently of one other. A few days later, when the connections between neurons are more mature, the dynamics of the system change completely. Episodes of global activity appear, characterized by the unison firing of the entire culture followed by silent intervals. These ‘bursting’ episodes are highly periodic and can last for several weeks.

Although spontaneous activity in cultures has been reported and studied for many years, little is known on the exact mechanisms that control the generation and maintenance of these episodes of global activity. Experiments show that these episodes are a collective effect that strongly depends on the properties of the culture and the connectivity between neurons.

Here we present a numerical model combined with experimental measurements to unders-tand the origins of SA. In our approach we consider the SA as a self-organized process, i.e. that neurons form connections and modify their strength until they are able to sustain episodes of SA with a given rate. We simulate realistically the way in which neurons grow and form connections to one another. This provides the connectivity matrix that defines the network and that mimics the one of the culture. Based on previous studies [2], we simulate the acti-vity of each neuron by combining the behaviour of its membrane potential with a dynamical coupling between neurons. Our model is able to reproduce the rhythmic SA episodes obser-ved experimentally and the role of neuronal connectivity. The model also provides a basis for understanding many recent observations, such as the presence of leader neurons or burst initiation zones.

[1] Eckmann, J.-P., Feinerman, O., Gruendlinger, L., Moses, E., Soriano, J., and Tlusty, T. (2007) Phys. Rep. 449, 54-76. [2] Alvarez-Lacalle, E., and Moses, E., (2009) J. Comput. Neurosci. 26, 475-493.

contactauthor:Javier G. ORLANDI

217


poster 110

nanomechanics of the tip-link cadherins and its role in hearingJavier Oroz1, Alejandro Valbuena1, Albert Galera-Prat1, Rubén Hervás1, Ulrich Müller2, & Mariano Carrión Vázquez1

1Instituto Cajal, CSIC, CIBERNED & IMDEA Nanociencia, Avda. Doctor Arce 37, 28002 Madrid2The Scripps Research Institute, 10550 North Torrey Pines Road, 92037 La Jolla, California.e-mail: [email protected].

The cochlear hair cells convert the deflexion of their stereocilia originated from auditory sti-muli into nervous signals [1]. A crucial element of this mechanotransduction, the transforma-tion of mechanical force into electrical signalling, is the tip-link, a structure located between stereocilia. This rigid system maintains tension between stereocilia allowing the opening of ionic channels due to the deflexion of the stereocilia, which generates a nervous stimulus [2]. Recently, it has been discovered that the tip link is made up by cadherin 23 and protocadhe-rin 15 heterophilically bound [3]. In this system, to know its response to mechanical forces is critical for our understanding of the molecular mechanism involved in its function. Here we present the nanomechanical analysis of cadherin 23 and protocadherin 15 ectodomains by single-molecule force spectroscopy based on Atomic Force Microscopy [4]. Similar to its paralog C-cadherin, the nanomechanics of the tip-link cadherins shows a tight regulation by calcium coordination, which mechanically stabilizes the ectodomains and further displaying an extramodular mechanical element (we call it “calcium rivet”) that can be ascribed to the rupture of the calcium coordination complexes [5]. Furthermore, the nanomechanical analysis of a pathological mutation related to the congenital deafness DFNB23 [3] reveals an alteration of the mechanical stability of the module carrying the mutation. We are also studying a mu-tation involved in DFNB12 [6]. Hence, our results suggest that these mechanical alterations could be the primary cause of the disease. This has to be tested in vivo. We finally present a descriptive model integrating our nanomechanical results on the pathological mutations in our current view of the hair cells [7].

[1] Ingber D (2006) FASEB J 20, 811-27.[2] Gillespie PG, Müller U (2009) Cell 139, 33-44.[3] Kazmierczak P, Sakaguchi H, Tokita J, Wilson-Kubalek EM, Milligan RA, Müller U, Kachar B (2007) Nature 449, 87-92. [4] Carrión-Vázquez M, Oberhauser AF, Díez H, Hervás R, Oroz J, Fernández J, Martínez-Martín D (2006) In Advanced Techniques in Biophysics, (Edited by Arrondo JLR, Alonso A), pp. 163-245. Springer-Verlag, Heidelberg.[5] Oroz J, Valbuena A, Vera AM, Mendieta J, Gómez-Puertas P, Carrión-Vázquez M (under review) PLoS Biology.[6] Schwander M, Xiong W, Tokita J, Lelli A, Elledge HM, et al. (2009) Proc Natl Acad Sci USA 106, 5252-7.[7] Shin J-B, Gillespie PG (2009) Proc Natl Acad Sci USA 106, 4959-60.

contactauthor:Javier OROZ

218

poster 111

Coupling lipid and protein composition to optimize surface activity of synthetic and semi-synthetic pulmonary surfactant preparationsOlga L. Ospina, David Schürch, Antonio Cruz and Jesús Pérez-Gil

Dept. Biochemistry, Fac. Biology, University Complutense, Madrid, Spain

Pulmonary surfactant is a lipid/protein complex secreted by alveolar type II cells in mamma-lian lungs, which principal function is to reduce the surface tension at the respiratory air/liquid interface and so preventing lung collapse at the end of expiration. Since the lack of or deficiencies in pulmonary surfactant are directly related with severe diseases such as the respiratory distress syndrome in preterm neonates, an important research effort has been directed to reveal the role of the different protein and lipid components in lung surfactant and to design and develop efficient surfactant preparations with potential clinical application.

Presence in surfactant of hydrophobic proteins SP-B and SP-C is important to provide to sur-face active lipids with an appropriate dynamic behaviour, including rapid interfacial adsorp-tion, stability of films subjected to compression, and efficient re-spreading during expansion. Although some of the activities of SP-B and SP-C seem to overlap, current models propose that the two proteins might cooperate to optimize interfacial surfactant performance. Attempts to develop synthetic or semi-synthetic clinical surfactants have proposed a variety of lipid compositions and surfactant protein-mimicking complements, and systematic studies are re-quired to determine the principles defining lipid and protein coupling as required to optimize surfactant performance.

In the present work we have compared the interfacial activity of surfactant preparations re-constituted from synthetic lipids and different proportions of surfactant proteins SP-B and/or SP-C in a captive bubble surfactometer (CBS). Two basic lipid mixtures have been tested. The ternary mixture DPPC/POPC/POPG (50:25:15), in the absence or in the presence of 5 or 10% cholesterol, roughly mimics the lipid composition of natural surfactant. Alternatively, DPPC/POPG/Palmitate mixtures are being widely used to prepare clinical surfactant preparations. We have assessed the ability of the different lipid/protein suspensions to i) rapidly adsorb at the bubble air-liquid interface, ii) stably produce very low surface tensions under quasi-static compression-expansion cycling and iii) maintain the lowest surface tensions with minimal compression and hysteresis under rapid physiological-like compression-expansion dynamics. Significant differences were found between the surface behaviour of surfactant preparations containing only SP-C or SP-B and preparations containing both proteins, once subjected to compression-expansion dynamics. Also, the efficient surface behaviour of the two assessed lipid mixtures was critically dependent on the presence of specific protein compositions, su-ggesting some defined dependences between protein and lipid moieties to simultaneously sustain mechanical stability and dynamics under physiologically-relevant constraints.

contactauthor:Olga L. OSPINA

219


poster 112

effect of hydrophobic surfactant proteins Sp-B and Sp-C on permeability of phospholipid membranesElisa Parra1, Lara H. Moleiro2, Antonio Alcaraz3, Iván López-Montero2, Antonio Cruz1, V.M. Aguilella3, Francisco Monroy2, Jesús Pérez-Gil1

1Dept. Biochemistry, Fac. Biology, UCM, Madrid.2Dept. Physical Chemistry, Fac. Chemistry, UCM, Madrid.3Dept. Physics, UJI, Castellón.

Pulmonary surfactant is a complex mixture of lipids and proteins whose main function is to reduce surface tension at the alveolar air–liquid interface of lungs in order to avoid alveolar collapse at the end of expiration and to facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific surfactant proteins, including the hydrophobic proteins SP-B and SP-C. In this study, we have analyzed the effect of hydrophobic surfactant proteins on the permeability of phospholipid membranes by using two different approaches: fluores-cence microscopy of giant vesicles and electroconduction in planar lipid membranes.

In the first case, two fluorescent water-soluble probes, FM®1-43 and calcein, have been asses-sed. The membrane-sensitive probe FM®1-43, which is non-fluorescent in water, only labels the external leaflet of membranes due to its amphiphilic character, and calcein emits green fluorescence in aqueous media. These properties allow us to study the effect of SP-B and SP-C on the structure and accessibility of membranes and aqueous compartments in giant POPC vesicles. In the presence of physiological amounts of both hydrophobic proteins SP-B and SP-C, giant oligolamellar vesicles incorporated almost instantaneously the probe FM®1-43 in every single membrane once added to the external medium. In contrast, oligolamellar vesicles made of pure POPC were only labelled in the outermost membrane layer. Lipid vesicles were impermeable for calcein, while this probe could permeate through membranes supplemented with SP-B, SP-C or mixtures of both proteins. Some differences were noticed between the effect of SP-B and SP-C on giant vesicles: suspensions containing only SP-B were stable but those containing only SP-C were quite dynamic, undergoing frequent fluctuations, reorgani-zations and ruptures as observed under the microscope. These results suggest that SP-B and SP-C could have different contributions to inter- and intra-membrane lipid dynamics.

On the other hand, planar lipid membranes (PLM) have been widely used to study ionic permeation through phospholipid bilayers mediated by membrane proteins. Permeability of phospholipid bilayers incorporating small amounts of SP-B, SP-C or both has been analyzed in PLMs prepared by the dual monolayer technique. The presence of either SP-B or SP-C in model planar bilayers allowed the passage of ions through model membranes; all the mea-surements carried out to date indicate the formation of a channel-like structure that can be characterized in terms of ionic conductance and selectivity. Possible implications of these structures in the biological context of the pulmonary surfactant system will be discussed.

contactauthor:Elisa PARRA

220

poster 113

effect of crowding by Dextrans on the hydrolysis of n-succinyl-L-phenyl-ala-p-nitroanilide catalyzed by alpha-chymotrypsinI. Pastor1, E. Vilaseca1, S. Madurga1, J. L. Garcés2, M. Cascante3 and F. Mas1

1Department of Physical Chemistry and Research Institute of Theoretical and Computational Chemistry (IQTCUB) of University of Barcelona (UB). Barcelona (Spain).2Department of Chemistry, University of Lleida (UdL), Lleida (Spain).3Department of Biochemistry and Molecular Biology and Institute of Biomedicine (IBUB) of University of Barcelona (UB) and IDIBAPS, Barcelona (Spain).

Traditionally, studies on the diffusion-controlled reaction of biological macromolecules have been carried out in dilute solutions (in vitro). However, in an intracellular environment (in vivo), there is a high concentration of macromolecules, which results in non-specific inte-ractions (macromolecular crowding). This affects the kinetics and thermodynamics of the reactions that occur in these systems. In this paper, we study the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroa-nilide catalyzed by alpha-chymotrypsin, by adding Dextrans of various molecular weights to the reaction solutions. The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction. A vmax decay and a Km increase were obtained when the Dextran concentration in the sample was increased. The increase in Km can be attributed to the slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in vmax could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media.

contactauthor:I. PASTOR

221


poster 114

Structural studies in mrna packaging machineryPena A1, Gewartowski K2, Montoya G3, Dziembowski A2 and Valpuesta JM1.1Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049, Madrid, Spain.2Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawinskiego 5a, 02-106 Warsaw, Poland.3Structural Biology and Biocomputing Programme, Macromolecular Crystallography Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain.

During transcription, the nascent pre-mRNA undergoes a series of processing steps, resulting in export-competent mRNA ribonucleoprotein complexes (mRNPs) that are transported to the cytoplasm [1]. Over the last years it has been shown that the different mRNA processing steps and the mRNP biogenesis and export processes are connected to each other and to transcription [2]. The THO complex was initially described as a four-protein complex that connects transcription/elongation with the incidence of mitotic recombination [3]. Further in-vestigation shows that THO is in fact, part of a larger complex termed TREX, which is involved in coupling mRNA transcription with export to the cytosol [4]. The TREX complex is required to coordinate events leading to the acquisition of export competent mRNPs [5] and may have a primary role in stable mRNP formation [6]. The THO complex has also been described as interacting with the Nucleotide Excision Repair machinery [7] and has been proposed to work in an mRNA quality control pathw ay at the transcription level through its interaction with the nuclear exosome [8].

The current view supports a model in which the THO/TREX complex travels along the active gene, interacting with the RNAPII and loading export factors into the nascent mRNA in a transcription-dependent manner [9]. The detailed mechanisms of how these events occur co-transcriptionaly remain unclear.

The THO/TREX complex has been described in S. cerevisae, D. melanogaster and Human [3, 4, 10]. So far, there is very little structural information on the THO/TREX complex.

In order to gain useful insights on the THO/TREX function, we have purified the yeast THO complex with the goal of studing its interaction with a the Tex1 protein, a component of the TREX complex whose exact function remains to be established. Using electron microscopy and image processing we have three-dimensionally reconstructed THO complex and we have also successfully determined the general shape of the complex, and the localization of the Tex1 protein interacting with the THO complex.

References:[1] R. Luna, H. Gaillard, C. Gonzalez-Aguilera, A. Aguilera, Chromosoma 117 (2008) 319-331.[2] M. Skruzny, C. Schneider, A. Racz, J. Weng, D. Tollervey, E. Hurt, PLoS biology 7(2009) e8.[3] S. Chavez, T. Beilharz, A.G. Rondon, H. Erdjument-Bromage, P. Tempst, J.Q.Svejstrup, T. Lithgow, A. Aguilera, The EMBO journal 19 (2000) 5824-5834.[4] K. Strasser, S. Masuda, P. Mason, J. Pfannstiel, M. Oppizzi, S. Rodriguez-Navarro,A.G. Rondon, A. Aguilera, K. Struhl, R. Reed, E. Hurt, Nature 417 (2002) 304-308.[5] M. Rougemaille, G. Dieppois, E. Kisseleva-Romanova, R.K. Gudipati, S. Lemoine,

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C. Blugeon, J. Boulay, T.H. Jensen, F. Stutz, F. Devaux, D. Libri, Cell 135 (2008)308-321.[6] D. Zenklusen, P. Vinciguerra, J.C. Wyss, F. Stutz, Molecular and cellular biology 22(2002) 8241-8253.[7] S. Gonzalez-Barrera, F. Prado, R. Verhage, J. Brouwer, A. Aguilera, Nucleic acidsresearch 30 (2002) 2193-2201.[8] S.A. Johnson, G. Cubberley, D.L. Bentley, Molecular cell 33 (2009) 215-226.[9] K.C. Abruzzi, S. Lacadie, M. Rosbash, The EMBO journal 23 (2004) 2620-2631.[10] J. Rehwinkel, A. Herold, K. Gari, T. Kocher, M. Rode, F.L. Ciccarelli, M. Wilm, E.Izaurralde, Nature structural & molecular biology 11 (2004) 558-566.

contactauthor:PENA A

223


poster 115

Molecular recognition of peloruside a by microtubules. the C24 primary alcohol is essential for biological activityBenet Pera1, Mina Razzak2, Chiara Trigili1, Oriol Pineda3, Angeles Canales1, Rubén M. Buey1, Jesús Jiménez-Barbero1, Peter T. Northcote4, Ian Paterson2, Isabel Barasoain1 and José Fer-nando Díaz1

1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Rami-ro de Maeztu 9, 28040 Madrid, Spain.2University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.3Departament de Química Orgànica, Facultat de Química, Universitat de Barcelona, Av. Dia-gonal 647, 08028 Barcelona, Spain.4Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand.

Peloruside A is a microtubule-stabilizing agent which targets the same or an overlapping site as laulimalide [1]. It binds to preformed microtubules with a 1:1 stoichiometry, with a binding affinity in the low µM range, reducing the number of microtubular protofilaments in the same

way as paclitaxel. Although the binding affinity of the compound is comparable to that of the low-affinity stabilizing agent sarcodictyin, peloruside A is more active in inducing microtubule assembly and is more cytotoxic to tumor cells, suggesting that the pelorusi-de site is a more effective site for stabilizing microtu-bules. In investigations towards the preparation of fluorescent-labelled peloruside derivates, two C24-acylated compounds were synthesized in order to asses this position as a useful handle for elaboration. Unlike the parent compound, they do not bind to preassembled stabilized microtubules and they not induce microtubule assembly in vitro. Despite this, the compounds were apparently active in cells, as they are hydrolyzed back to peloruside in the cell media and thus recover biological activity. According to molecular modeling, this substitution at the C24 hydroxyl group presumably disrupts the interaction of the side chain with Arg320 in the putative binding

site on α-tubulin [2]. The binding epitope of peloruside A on microtubules has been determi-ned using NMR techniques, being compatible with the α-tubulin binding site.

[1] Gaitanos, T.N., Buey, R.M., Díaz, J.F., Northcote, P.T., Teesdale-Spittle, P., Andreu, J.M. and Miller, J.H. (2004) Cancer Res. 64, 5063-5067.[2] Jiménez-Barbero, J., Canales, A., Northcote, P.T., Buey, R.M., Andreu, J.M., and Díaz, J.F. (2006) J. Am. Chem. Soc. 128, 8757-8765.

contactauthor:Benet PERA

Model of the interaction of peloruside A (blue-red) and C24-acylated compound (yellow-red) in the peloruside binding site propose in α-tubulin.

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poster 116

Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomesJosé M de Pereda1, Pilar Lillo2, Arnoud Sonnenberg3

1Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas – Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain.2Departamento de Biofísica, Instituto de Química Física Rocasolano. Consejo Superior de Investigaciones Científicas, Serrano 119. 28006 Madrid, Spain.3Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Hemidesmosomes are adhesive protein complexes that mediate stable attachment of basal epithelial cells to the basement membrane by connecting the extracellular matrix to the in-termediate filament cytoskeleton. The integrin α6β4 (a laminin receptor) and the cytoske-letal protein plectin are key components of the hemidesmosomes. Binding of the cytoplasmic moiety of integrin β4 subunit to plectin is essential for the formation and stability of hemi-desmosomes. In order to better understand the molecular recognition mechanisms responsi-ble for the assembly and stability of hemidesmosomes, we have solved the crystal structure of the integrin β4-plectin primary complex at 2.75 Å resolution. The complex is formed by the first pair of fibronectin type III (FnIII) domains of integrin β4 and the actin binding domain (ABD) of plectin. The largest area of contact occurs between the second FnIII domain of β4 and the first calponin homology (CH1) domain of the ABD of plectin. Two basic residues in the second FnIII of β4, R1225 and R1281, which are mutated in patients suffering from non-lethal forms of the blistering disease epidermolysis bullosa, establish essential contacts with plectin. We have also solved the crystal structure of the aforementioned region of integrin β4 in a free form. Comparison of the free and plectin-bound structures reveals a reorganization of the segment downstream from the second FnIII sequence upon binding to plectin. By using a β4 mutant that can be blocked in the free conformation we have shown that the conforma-tional change in β4 is required for optimal binding, suggesting that the interaction between β4 and plectin may be subject to allosteric regulation.

contactauthor:José M de PEREDA

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poster 117

Modelling the F1-atpaseRubén Pérez

The cellular environment is an out of equilibrium system in which there is a continuous trans-duction of matter and energy. The mechanisms driving this processes are the molecular mo-tors. There are many different Molecular Motors with many different functions. For instance, kinesin, which can transport cargoes along microtubule tracks along the cell; or BFM, which rotates the flagella of bacteria allowing them to propel in the extracellular media.

What is even more interesting is that Molecular Motors are able to achieve its goal despite the high noise media in which the live. The origin of this fluctuations are two: Thermal fluc-tuations, which are comparable to typical energies of Molecular Motors; and the discreteness of molecules in the system.

One particular molecular motor is the F1-ATPase, part of the ATPsyntase holoenzyme. F1-ATPase is a rotatory motor that uses the hydrolysis energy of ATP to rotate its central shaft. F1-ATPase is reversible and can also synthesize ATP out of its hydrolysis products when the shaft is mechanically rotated in the proper direction. The rotation trajectories can be expe-rimentally studied by attaching a load to the shaft. The aim of our work is to analyze such trajectories studying the mechanico-chemical properties of the motor and the biochemical information available.

The resulting model is a flashing ratchet model able to reproduce the stepping trajectories observed. The model returns easy analytical expressions for the average velocity of the motor that compare accurately with the dependence of the velocity on external parameters such as the ATP concentration, the friction of the load, ATP hydrolysis energy or thermal fluctuations. The model is also the first model able to predict the complete substep phenomenology obser-ved in experiments, obtaining and universal value for the substep angle.

R. Perez Carrasco and J.M. Sancho, Modelling the F1-ATPase, Biophysical Journal, (2010) (Accepted)

contactauthor:rubénPéREZ

226

poster 118

Biophysical studies reveal weak points in the adenovirus capsidPérez-Berná AJ1, Menéndez-Conejero R1, Menéndez M2, Flint SJ3 and San Martín C1.1Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain2Instituto de Química Física Rocasolano (IQFR-CSIC), Madrid, Spain3Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

The human adenovirus type 2 ts1 mutant, stalled at the immature state, does not package the viral protease and produces capsids containing unprocessed protein precursors. Ad2 ts1 grown at the non-permissive temperature is not infectious. It has been shown that the defect in infectivity is linked to a defect in uncoating [1]. We have previously described how the precursor proteins act as scaffolding elements hyperstabilizing both the immature capsid and core [2].

In this work we compare the uncoating behavior of immature ts1 and wildtype virus. De-naturing agents, as well as changes in pH, temperature, or ionic strength, cause changes in the secondary, tertiary, and quaternary structure of the viral proteins. Differential scanning calorimetry and extrinsic fluorescence have been used to monitor these changes. Electron mi-croscopy helps to correlate the biophysical data with the structural changes that occur during viral disassembly, and to identify conditions that alter the structural integrity of adenovirus capsid.

The immature and mature viruses present large differences in their disassembly behavior. Ad2 ts1 displays higher stability than the wildtype virus in all the disruption assays, in agre-ement with its uncoating phenotype. Interestingly, different kinds of stress result in different rupture patterns. In heat disruption experiments, the mature virus completely disassembles following and “all or nothing” process, while ts1 releases pentamers. On the other hand, high ionic strength or low pH result in random capsid fractures across hexameric positions for both mature and immature virus. Thus, our work experimentally reveals two modes of shell bursting for a T=25 icosahedral capsid, consistent with previously proposed models for ther-modynamic and mechanical failure [3].

[1] Weber, J.M. (1999) Role of endoprotease in adenovirus infection. In Seth, P. (ed.), Adenoviruses : basic biology to gene therapy. R.G. Landes, Austin, Tex., U.S.A., pp. 79-83.[2] Pérez-Berná AJ, Marabini R, Scheres SH, Menéndez-Conejero R, Dmitriev IP, Curiel DT, Mangel WF, Flint SJ, San Martín C. Structure and uncoating of immature adenovirus. J Mol Biol. 2009 Sep 18;392(2):547-57. .[3] Zandi, R. and Reguera, D. (2005) Mechanical properties of viral capsids. Phys Rev E Stat Nonlin Soft Matter Phys, 72, 021917.

contactauthor:PéREZ-BERNÁ, AJ

227


poster 119

the three-dimensional characterization of the DnaK:grpe complexMaría Angeles Pérez-Calvo1, Judit Perales2, Fernando Moro2, Oscar Llorca3, Arturo Muga2 and José María Valpuesta1

1Centro Nacional de Biotecnología - CSIC. Madrid, Spain.2Unidad de Biofísica. Universidad del País Vasco - CSIC. Bilbao, Spain.3Centro de Investigaciones Biológicas - CSIC. Madrid, Spain.

Molecular chaperones are essential for the appropriate folding of proteins in the cell [1]. DnaK, the main Hsp70 (Heat shock protein 70kDa) protein in bacteria, is a molecular chape-rone with ATPase function [2]. This chaperone also regulates many processes, like apoptosis or DNA replication [3]. This versatility is achieved in part through the interaction with other cochaperones like Hsp40s and nucleotide exchange factors (NEFs), which regulate the acti-vity of DnaK [4].

DnaK functions through successive rounds of unfolded protein binding and release. Cycling between high and low affinity states for substrates is regulated by a nucleotide-induced allos-teric transition between the nucleotide (NBD) and substrate (SBD) binding domains that is controlled by cochaperones. One of these cochaperones, the NEF GrpE accelerates nucleotide exchange [5].

The aim of this work is to characterize the interaction between DnaK and its nucleotide ex-change factor GrpE. A crystallographic structure of a complex between GrpE and DnaK NBD has already been published, which nevertheless has not been able to show in full the interac-tion between the chaperone and its cofactor [6]. We have three-dimensionally reconstructed the complex formed between the full-length DnaK and GrpE by electron microscopy. The structure suggests a more complex interaction between the two proteins since it reveals that both domains of DnaK embrace the head of GrpE. Our kinetics experiments with a peptide substrate confirm an interaction of the unstructured region of the GrpE tail with the DnaK SBD acting as a pseudosubstrate, as it has been already described. These experiments also show an increase of the affinity for the substrate when DnaK is complexed with GrpE. All these results could be explained by the DnaK SBD interacting with GrpE regions other than the disordered N-terminal domain; thus suggesting that GrpE, besides being a nucleotide exchange factor, has a role in DnaK-peptide binding.

[1] Popp, S., Packschies, L., Radzwill, N., Vogel, K.P., Steinhoff, H. and Reinstein, J. (2005) J. Mol. Biol., 347, 1039–1052 [2] Moro, F., Taneva, S.G., Velázquez-Campoy, A. and Muga, A. (2007) J. Mol. Biol., 374, 1054-64 [3] Peres Ben-Zvi, A. and Goloubinoff, P. (2001) J. of Structural Biology, 135, 64-93 [4] Bukau, B., Weissman, J. and Horwich, A. (2006) Cell, 125, 443-451[5] Sousa, R. and Lafer, E.M. (2006) Traffic, 7, 1596-1603 [6] Harrison, C.J., Hayer-Hartl, M., Di Liberto, M., Hartl F.U. and Kuriyan, J. (1997) Science, 276, 431-435

contactauthor:María Angeles PéREZ-CALVO

228

poster 120

the Cp43´ complex of the cyanobacterium Synechocystis pCC 6803 under Fe-starvation.R. Picorel1, M.A. Luján1, P. Llorente1, R. Cases2, M. Seibert3 and R. Jankowiak4.1Estación Experimental de Aula Dei (CSIC), Avda. Montañanan 1005, E-50059 Zaragoza, Spain. 2University of Zaragoza-ICMA, C/ Cerbuna 12, E-50009 Zaragoza, Spain. 3National Renewable Energy Laboratory (NREL), Cole Boulevard 1617, Golden CO 80401, USA. 4Kansas State University; Manhattan KS 66502, USA.

The cyanobacterium Synechocystis PCC 6803 under Fe-starvation induces the isiA gene that encodes the photosynthetic CP43´pigment-protein complex. This complex surrounds the PSI core trimer forming a ring of 18meres. This PSI-CP43´supercomplex is very stable and allows efficient energy transfer. The CP43ćomplex is very similar to the CP43 complex of the PSII proximal antenna but its precise function is under debate. One of the most cited hypotheses describes the CP43ćomplex as a main antenna to funnel excitation energy to PSI avoiding, thus, PSII photoinhibition under stress conditions. In this work, we describe the isolation of the CP43ćomplex and some spectroscopic features in comparison to the PSI-CP43´supercomplex. The supercomplex was isolated from the wild type organism and the CP43ćomplex from psaFJ, a PSI defective mutant. Thylakoid membranes were solu-bilised with the detergent n-dodecyl β-D-maltoside (β-DM) and the extract subjected to one differential centrifugation, one sucrose gradient centrifugation, two DEAE chromatogra-phies, and one Q-Sepharose chromatography. The quality of the preparations was assessed by SDS-PAGE, UV-VIS and fluorescence spectroscopy. A pure supercomplex showed two main protein bands at around 65 and 36 kDa, which correspond to PSI-core and CP43´proteins, respectively. Pure CP43ćomplex showed a single main protein band at around 36 kDa. The PSI-CP43ánd the CP43ábsorbed at 673 and 670.5 nm, respectively, while the fluores-cence emission spectra of PSI-CP43´displayed two main peaks at 685 and 722 nm, and the CP43ćomplex a single peak at 685 nm. Less quality CP43´samples showed some emission over 700 nm, most probably due to highly fluorescent contaminant phycobilines. Low-tem-perature UV-VIS spectroscopy revealed the presence of several groups of chlorophylls (Chl) due to different protein-Chl and Chl-Chl electronic interactions. Moreover, the absorption and fluorescence peak maxima underwent a blue-shift as a function of time and intensity of the hole-burning laser.

contactauthor:R. PICOREL

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poster 121

physiological splice variant of hMgCS2 geneMónica Ramos1, Eduardo López-Viñas2,3, Beatriz Puisac1, María Arnedo1, Sebastián Menao1, María Concepción Gil-Rodríguez1, María Esperanza Teresa-Rodrigo1, Angeles Pié1, Feliciano J. Ramos1, Paulino Gómez-Puertas2, Juan Pié1. 1Laboratory of Clinical Genetics and Functional Genomics. Departments of Pharmacology-Physiology and Pediatrics, Medical School, University of Zaragoza, Zaragoza, Spain.2Molecular Modelling Group, Center of Molecular Biology “Severo Ochoa” (CSIC-UAM), Cantoblanco, Madrid, Spain and “CIBER-Obn Physiopathology of Obesity and Nutrition” (CB06/03/0026), Instituto de Salud Carlos III, Spain.3Biomol-Informatics SL. c/ Faraday, 7. Parque Científico de Madrid. Cantoblanco. 28049 Madrid, Spain.

The HMGCS2 gene encodes a key enzyme in the regulation of ketogenesis, the human mito-chondrial HMG-CoA synthase (mHS). This enzyme catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). It has been des-cribed regulatory mechanisms of this enzyme at both transcript and protein level. However, despite the increasing importance of splicing as a mechanism of proteome expansion and of regulation of gene expression, it has not been studied before the physiological and patholo-gical splice variants of HMGCS2 gene. The aim of this study was to identify potential splice variants of this gene and to analyze how the structure of the enzyme may be affected.

To identify alternative transcripts, it has been used a strategy of amplification of cDNA in overlapping fragments. From cDNA amplification, it has been detected a transcript with loss of the complete exon 4. The produced protein from this variant, although maintained the reading frame, lost 55 aminoacids.

To locate splicing variants in the structure of mHS, a 3D model of the protein was initially generated through standard homology modelling techniques using the Swiss-PdbViewer pro-gram and the Swiss-Model server, selecting as template the structure of the human cytosolic HMG-CoA synthase (Turnbull et al., 2007).The homo-dimer modelled construction was suc-cessfully validated later by the published crystal structure of mHS (Yue 2008). Both modelled and crystallized mHS shows that the deletion of exon 4 results in loss of β8 sheet and α7 helix. This deletion affects a core area of the protein close to the substrate locus and to the interaction surface between both protein monomers. These results suggest that the resulting protein would be unstable and devoid of enzyme activity.

contactauthor:Juan PIé

230

poster 122

three dimensional structure model of the nipBL heat repeatsMaría Concepción Gil-Rodríguez1, Eduardo López-Viñas2,3, Beatriz Puisac1, María Arnedo1, María Esperanza Teresa-Rodrigo1, Milagros Ciero1, Angeles Pié1, Feliciano J. Ramos1, Paulino Gómez-Puertas2, Juan Pié1.1Laboratory of Clinical Genetics and Functional Genomics. Departments of Pharmacology-Physiology and Pediatrics, Medical School, University of Zaragoza, Zaragoza, Spain.2Molecular Modelling Group, Center of Molecular Biology “Severo Ochoa” (CSIC-UAM), Cantoblanco, Madrid, Spain and “CIBER-Obn Physiopathology of Obesity and Nutrition” (CB06/03/0026), Instituto de Salud Carlos III, Spain.3Biomol-Informatics SL. c/ Faraday, 7. Parque Científico de Madrid. Cantoblanco. 28049 Madrid, Spain.

Mutations in NIPBL, a regulator of the Cohesin complex, were the first demonstrated cau-se of Cornelia de Lange Syndrome (CdLS, MIM 122470). CdLS manifests facial dysmorphic features, growth and cognitive impairment, and limb malformations. Recently, mutations in the Cohesin structural components SMC1A and SMC3 were found in patients with atypical CdLS. The physical and functional interactions between these elements of the Cohesin ring are currently poorly defined. Helical folds of HEAT repeats have been proposed in NIPBL as a potential site of interaction with other proteins, but no crystal structure for NIPBL has been solved to date.

Analysis of NIPBL sequence features using the Pfam and Uniprot databases indicated the presence of HEAT repeat elements spanning residues 1767-2351. Since three dimensional structures of some HEAT repeats-containing proteins have been experimentally obtained, we used a position-specific scoring matrix-based structure prediction method, Phyre (the newest version of 3D-PSSM) to obtain a sequence-to-structure alignment to build a model for the mul-tiple HEAT domains in NIPBL. The top ten structures compatible with HEAT repeats predicted a NIPBL domain that belonged to the ARM/HEAT repeat structural superfamily composed of a superhelix of alpha helices. The largest sequence coverage and percentage of sequence identity (NIPBL residues 1538 to 2242) was to the SCOP template c1u6gc, corresponding to the crystal structure of human TATA binding protein TIP120 in complex to cullin-1 and ring-box protein-1 (PDB code: 1UG6) with an E-value of 8.0e-4 and 95% of internal estimated pre-cision of structure prediction. Interestingly, in second threading round, the predicted HEAT region was further extended to residue 2470. A three-dimensional model was then built for the predicted HEAT region, showing the characteristic super-helix fold composed of pairs of anti-parallel alpha helices connected by unstructured loops of short to medium length.

Interestingly, this region is clearly affected by missense mutations, indicating that the structu-re of the HEAT domain plays a key role in the functionality of the protein. Missense mutations could prompt local variations in protein interactions, either between pairs of alpha-helices, or between the HEAT domain and its putative accompanying macromolecules. Further analyses will be necessary to identify putative interacting proteins and the physiological role of any interaction.

contactauthor:Juan PIé

231


poster 123

Structural characterization of protein-protein complexes by integrating computational docking with small-angle scattering dataCarles Pons1,2 Marco D’Abramo3,4 Dmitri I. Svergun5, Modesto Orozco2,3,4,Pau Bernadó6, Juan Fernández-Recio1

1Barcelona Supercomputing Center (BSC), Barcelona 08034, Spain2Computational Bioinformatics, National Institute of Bioinformatics (INB), Barcelona 08034, Spain3Joint IRB-BSC Program on Computational Biology, Institute for Research in Biomedicine, Parc Científic de Barcelona, Barcelona 08028, Spain4Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona 08028, Spain5European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany.6Institute for Research in Biomedicine, Parc Científic de Barcelona, Barcelona 08028, Spain

X-ray crystallography and NMR can provide detailed structural information of protein-pro-tein complexes, but their high-throughput application is challenging. Other methods such as Small-Angle X-ray Scattering (SAXS) are more promising for large-scale application, but at the cost of lower resolution. Fortunately, protein-protein docking algorithms can complement available experimental information at low resolution[1]. In this study we propose a novel stra-tegy that combines SAXS data[2] and theoretical protein-protein docking[3]. This approach has been tested on a large benchmark of known structures with synthetic SAXS data, and on three experimental examples. The combined approach (pyDockSAXS) provided better suc-cess rate (43% for the top 10 predictions) than the two methods when applied alone, which clearly showed that binding energy can drive and complement the evaluation of experimental SAXS data. Further analyses according to different parameters made possible to dramatically increase success rates for specific groups of cases, and to define guidelines to improve data driven protein-protein docking.

[1] D’Abramo, M., Meyer, T., Bernadó, P., Pons, C., Fernández-Recio, J., and Orozco, M. (2009). On the Use of low-resolution Data to Improve Structure Prediction of Proteins and Protein Complexes. J. Chem. Theory Comput 5, 3129-3137.[2] Svergun, D., Barberato, C., and Koch, M.H.J. (1995). CRYSOL – a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates. J. Appl. Crystallogr 28, 768-773.[3] Cheng, T.M., Blundell, T.L., and Fernandez-Recio, J. (2007). pyDock: electrostatics and desolvation for effective scoring of rigid-body protein-protein docking. Proteins 68, 503-515.

contactauthor:Carles PONS

232

poster 124

probing the interaction between divalent cations and ompF channel residues: inversion of ionic selectivity and pKa shift of acidic residuesMaría Queralt-Martín, Elena García-Giménez, M. Lidón López, Antonio Alcaraz

Laboratory of Molecular Biophysics. Department of Physics. University Jaume I, Castellón, Spain.

In contrast to the highly-selective channels of neurophysiology that mostly employ the exclu-sion mechanism, quite different factors account for the selectivity of large channels. Elucida-tion of these factors is essential for understanding large channel selectivity and its regulation in vivo. The interaction between divalent cations and a protein channel, the OmpF porin has been investigated paying attention to the channel selectivity and its dependence with solution pH. From selectivity performed at neutral pH, we find that the moderate cationic selectivity of the general bacterial porin OmpF in sodium and potassium chloride solutions is reversed to anionic selectivity in salts of divalent cations (Ca2+, Mg2+, Ba2+, Ni2+). To understand the origin of this phenomenon we consider several factors which include binding of diva-lent cations, electrostatic and steric exclusion of differently charged and differently sized ions, size-dependent hydrodynamic hindrance, electrokinetic effects, and significant “anionic” diffusion potential for bulk solutions of chlorides of divalent cations. Though all these factors contribute to the measured selectivity of this large channel, the observed selectivity inversion is mostly due to the following two: first, binding of divalent cations compensates, or even slightly overcompensates, the negative charge of the OmpF protein, which is known to be the main cause of cationic selectivity in sodium and potassium chloride solutions; second, the higher anionic (vs. cationic) transport rate expected for bulk solutions of chloride salts of divalent cations is the leading cause of the measured anionic selectivity of the channel. The interaction of divalent cations with the channel has an additional interesting effect: the anionic selectivity found in salts of divalent cations is barely sensitive to the solution pH. This suggests that the binding of divalent cations could yield pKa values of ionizable groups signifi-cantly different than those of the isolated groups in solution. A simple molecular model based on statistical thermodynamics provides qualitative explanations to the experimental findings and can be useful for future, more elaborated treatments.

contactauthor:María QUERALT-MARTíN

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poster 125

Salt-dependent ph sensing and cooperative transport in a protein ion channel revealed by current noise, conductance and selectivity experimentsMaría Queralt-Martín, Elena García-Giménez, Vicente M. Aguilella, Antonio Alcaraz

Laboratory of Molecular Biophysics. Department of Physics. University Jaume I, Castellón, Spain.

The concept of positive cooperativity appeared in the study of oxygen uptake by hemoglobin to explain that when a molecule of oxygen binds makes it easier for a second molecule to bind. Quite the reverse, negative cooperativity refers to the situation where the presence of the first molecule makes it more difficult for the second molecule to bind. We study here the effect of salt on the pH titration of the OmpF channel, paying attention to the current noise, conductance and ion selectivity that are analyzed in terms of the Hill formalism. In all cases, Hill coefficients lower than 1 are found, suggesting a negative cooperativity. Although OmpF porin is a trimer, it was shown by a number of different methods that each monomer is iden-tical and functionally independent. Thus, the slowed-down channel titration is a property of each monomer. Surprisingly, we find that increasing salt concentration promotes negative cooperativity, which is seen as a salt-induced decrease of the Hill coefficient. This observation seems to exclude direct electrostatic interactions between protonation sites as the source of the phenomenon, suggesting another, more subtle mechanism(s). The experiments with the D113C/E117C mutant show the crucial role of these two acidic residues. Temperature de-pendent experiments also suggest that the negative cooperativity observed has probably two sources: the spurious cooperativity coming from the successive independent protonation of a large number of residues and the genuine cooperativity due to the real interaction between D113 and E117. Such interaction involving D113 and E117 residues has no noticeable effects at neutral pH, but has a critical effect at low pH because results in an inhibition of channel conductance that additionally provides an anionic selectivity to the channel. This suggests that the binding site could play a certain role in the protection of the bacteria against acidic media.

contactauthor:María QUERALT-MARTíN

234

poster 126

nucleoplasmin binds histone h2a-h2b dimers through its distal faceIsbaal Ramos1,2,*, Jaime Martín-Benito3,*, Ron Finn4 , Laura Bretaña1,2, Kerman Aloria5, Jesús M Arizmendi1,5, Juan Ausió4, Arturo Muga1,2, José M Valpuesta3 and Adelina Prado1,2.1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología. Uni-versidad del País Vasco. 48080 Bilbao, Spain2Unidad de Biofísica (CSIC-UPV/EHU), Aptdo. 644, 48080 Bilbao3Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Darwin, 3, 28049 Madrid, Spain4Department of Biochemistry and Microbiology. University of Victoria. Victoria, BC V8W3P6, Canada 5Servicio General de Proteómica-SGIker (ProteoRed). Universidad del Pais Vasco. 48080 Bilbao, Spain

* These authors contributed equally to this work.

Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chro-matin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B his-tone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers, and that the amount of bound ligands depends on the overall charge (phosphorylation level) of the cha-perone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information helps to unders-tand the function of NP as a histone chaperone.

contactauthor:Isbaal RAMOS

235


poster 127

Metallic cations modulate the nanomechanical response of phospholipid model membranes. a force spectroscopy study.L. Redondo-Morata1, 2, 3, G. Oncins4 and F.Sanz1, 3, 2 1Institute for Bioengineering of Catalonia (IBEC)2CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)3Department of Physical Chemistry,University of Barcelona 4Scientific Technical Services UB (SCT-UB).

How do metal cations affect the stability and structure of phospholipid bilayers? Which role does ion binding play in the insertion of proteins and in the overall mechanical stability of bio-logical membranes? To characterize such effects, several theoretical and microscopic appro-aches have been proposed in the past to study the mechanical properties of lipid bilayers. While providing crucial information, molecular dynamics simulations can not completely deal with the extraordinary complexity of biological membranes. Experimental techniques also have problems when it comes to test ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new perspective from the nanometric scale, where most of the specific molecular phenomena are taking place. Atomic Force Microscopy has sprung up as an essential tool to examine the lipid bilayers structure and behaviour.

The role of divalent cations was long known to have an effect on the packing properties of lipid bilayers. By contrast, the role of monovalent ions on the bilayer structure has been for a long time underestimated. Despite this prevailing view, recent molecular dynamicssimula-tions showed monovalent cations such as Na+ [1], would have an important effect on bilayer packing. From the experimental point of view, Garcia- Manyes et al. [3] has reported the effect of the ionic strength in the mechanical properties of model membranes, showing that the deposition process ant the mechanical stability of the lipids are highly dependent on the electrolyte solution.

Here we use force spectroscopy to quantitatively characterize the nanomechanical resistance as a function of the electrolyte concentration and composition thanks to a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systema-tically testing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent cations, we provide a wealth of information which unambiguously proves an independent and important contribution of each ion to the gross mechanical resistance, reporting quantitative measurements for membrane elastic modulus and also for its plastic properties. This work deals with the need of assessing the effect of different ions in the structure of phospholipid membranes, opening up new avenues for cha-racterizing themembrane (nano)mechanical stability.

[1] R.A. Bockmann, A. Hac, T. Heimburg, H. Grubmuller (2003), Biophys. J. 85, 1647–1655.[2] S. Garcia-Manyes, G. Oncins, F. Sanz, (2005) Biophys. J. 89, 1812–1826.

contactauthor:L. REDONDO-MORATA

236

poster 128

immobilization and characterization of Kcsa potassium channel into sol-gel glassesRocío Esquembre, María L. Renart, José A. Poveda, José M. González-Ros and C.Reyes Ma-teo.

Instituto de Biología Molecular y Celular. Universidad Miguel Hernández de Elche03202 Elche (Alicante), Spain.

Ion channels are membrane proteins involved in the maintenance of the appropriate ion ba-lance across the biological membrane, connecting the inside of the cell to its outside in a selective fashion. The ability to immobilize these proteins in inorganic matrices represent a significant step forward in developing a new generation of biologically active materials with potential applications in areas such as high throughput drug screening and for new genera-tion of sensors. [1]

The major problems limiting the immobilization of the lipid membrane-ion channel systems are their lower stability, as well as the necessity to develop a methodology able to retain the physical properties of the lipid bilayer, since it is the media where membrane proteins per-form their activity.

One of the most successful immobilization method for biological systems is the encapsula-tion in silica matrices prepared using the sol-gel process. But to date, the sol-gel technology has focused mainly on soluble proteins but not on membrane proteins. Recently, we used commercially available silica matrices precursors by means of an alcohol-free sol-gel route to successfully immobilize the transmembrane polypeptide gramicidin. This study shown that the gramicidin ion channel forming peptide retained its conformation and activity upon en-capsulation.[2] This was the first stage for immobilization works with bigger and more clini-cally relevant membrane proteins. Here we use the same experimental protocol to immobilize the transmembrane ion channel KcsA in a sol-gel matrix. KcsA was purified, reconstituted in DOPE:DOPG (7:3) liposomes, and the effects of sol-gel immobilization on the structure of KcsA and its ability of still suffering conformational changes after entrapment (due to interaction with conducting and blocking ions or different pH) were determined from changes in the photophysical properties of its tryptophan residues (fluorescence spectra and lifetimes) and quenching experiments. The thermal stability of the protein was also tested by following changes in the intrinsic fluorescence of the channel. In addition, the physical state of the lipid membrane was analysed by measuring the spectral shift of the fluorescent probe laurdan.

[1] Dunn, B., Zink, J.I., Accounts of Chemical Research, 40 ,9, (2007) 747.[2] Esquembre, R., Poveda, J.A., R., Mateo, C.R, Journal of Physical Chemistry B 113 (21), pp. 7534-7540, 2009

contactauthor:C. REYES MATEO

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poster 129

iL-1β effect on gLut5 intestinal transporterAlberto García-Barrios1, 3, Natalia Guillén2, 3, Sonia Gascón1, 3, Jesús de la Osada2, 3, Isabel Escu-dero1, Carmen Viñuales1, Mª Carmen Rodríguez-Yoldi1, Mª Jesús Rodríguez-Yoldi 1, 3.1Physiology Unit. Dept. of Pharmacology and Physiology2Biochemistry Unit. Dept. of Biochemistry and Molecular Biology. Veterinary Faculty. Universi-ty of Zaragoza. 50013 Zaragoza. Spain. 3CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III (ISCIII), Spain.

Interleukin-1β (IL-1β) is a pleiotropic cytokine produced by cells of the immune system and a large variety of other cell types including endothelial cells. It is released during inflammatory and infectious diseases, and possesses a wide spectrum of autocrine, paracrine and endocrine activities. The aim of this work was to examine the IL-1β effect on GLUT5 intestinal transpor-ter. A sepsis condition was induced, in rabbits, for 90 min after intravenous –iv- administra-tion of IL-1β 0.2 μg/kg body weight –bw-. Intestinal fructose transport can occur via GLUT2 and GLUT5 facilitative transporters and depends on the sugar concentration gradient across the membrane. Changes in fructose uptake might therefore result from changes in transpor-ter abundance in the plasma membrane and/or from metabolic alterations by increasing the slope of sugar gradients [1]. In this study, we performed fructose uptakes in purified brush border membrane vesicles (BBMV) of rabbit jejunum. Sugar uptake was decreased up to 40% at 10 min of incubation time in BBMVs prepared from IL-1β compared with control animals. To decipher the respective contribution of GLUT2 and GLUT5 to fructose transport, BBMVs were incubated for 10 min with 0.1 mM cytochalasin B, a competitive inhibitor of GLUT2 but not GLUT5. Fructose uptake was not changed in control and IL-1β BBMVs, by cytochalasin B, indicating the absence of GLUT2 contribution. In the same way, the cytokine did not inhibit D-fructose uptake when 50 mM D-fructose (acting as an inhibitor of mediated transport) was added to media. All together these results indicate that Il-1β-sensitive fructose transport is by GLUT5 at the level of the BBM excluding any effect of metabolism. Moreover, the levels of GLUT5 were same in all animal groups, indicating that the cytokine, at the time assayed, induces a reduction in the relative intrinsic activity of GLUT5 protein. On the other hand, IL-1β increased GLUT5 mRNA levels. Several mechanisms could be involved in this discrepancy, first it can not be ruled out a different time-course of mRNA and protein levels, second a post-transcriptional regulation of mRNA and third a regulatory effect at the GLUT5 cargo to be directed towards brush-border membrane. Acknolegment: This work was supported by grants from FIS and ISCIII CB06/03/1012, Dept. de Ciencia, Tecnología y Universidad del Gobierno de Aragón (Spain): A.32, PM051/2007 and PI017/09. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT) BFU2007-30688-E/BFI).

[1] Leturque, A., Brot-Laroche, E., Le Gall, M. (2009) Am. J. Physiol. Endocrinol. Metab. 296: E985-E992.

contactauthor:RODRíGUEZ-YOLDI

238

poster 130

Structural Characterization of the Centrosomal protein na14Rodríguez-Rodríguez, M.1, Treviño, M.1, Laurents, D.V.1, Arranz, R.2, Rico, M.1, Bruix, M.1 and Jiménez, M.A.1

1Dep. de Química-Física Biológica, Instituto de Química-Física Rocasolano, CSIC. 2Dep. de Estructura de Macromoléculas. Centro Nacional de Biotecnología, CSIC.

NA14 was initially identified as a minor autoantigen recognized by an autoimmune serum from a Sjögren syndrome patient. This 119 residues protein is restricted to the nucleus [1]. Analysis of the NA14 amino acid sequence with the Coil program showed that a significant portion of the protein has a high probability of forming helix (residues 6-80). According to this analysis, the protein is composed of an acidic coiled-coil domain pI=4.74 and a C-terminal basic domain pI=10.22. Furthermore, a sequence motif similar to that of leucine zippers in eukaryotic transcription factors is found in the segment spanning residues 8 to 22. This motif promotes dimerization or oligomerization through the formation of α-helical coiled-coil. Mo-reover, Errico et al. [2] suggest that NA14 and spastin interact in vivo and they propose that NA14 is a molecular adaptor involved in targeting spastin to the centrosome. Remarkably, the specific role of NA14 at the centrosome is completely unknown though some authors hypo-thesize that DIP13 and NA14 may represent a conserved protein family involved either in a general stabilization of microtubules or in linking microtubular structures to motile systems [3].

In this work, NA14 and some variants have been expressed in E. coli as His-tagged recombi-nant proteins. In all cases, the expression resulted in the formation of inclusion bodies and the purification by Ni-NTA-agarose was performed in the presence of 8 M urea. Afterwards, pro-tein samples were extensively dialyzed against the appropriate buffer with or without DPC. In order to characterize the protein structure we have used a variety of biophysical methods. These include secondary structure determination by CD spectroscopy, solubility by absorban-ce spectroscopy, presence of folded structure by nuclear magnetic resonance and imaging by electron microscopy. Furthermore, the extent of aggregation of NA14 was determined by size exclusion chromatography in different conditions and by analytical ultracentrifugation. All studies suggest that the protein forms self-associating helical fibers. We have also studied the conformational stability and the thermodynamic parameters which describe the folding transitions of NA14.

These results will increase our knowledge on the mechanisms underlying NA14 stability and fiber formation as well as their relation with the physiological and pathological processes involving centrosomal associated proteins.

[1] Ramos-Morales, F., Infante, C., Fedriano, C., Bornens, M. and Rios R.M. (1998) J. Biol. Chem. . 273, 1634-1639.[2] Errico, A., Claudiani, P., D`Addio, M. and Rugarli E. I. (2004) Human Mol. Gen. 13, 2121-2132. [3 Pfannenschmid F., Wimmer, V. C., Ríos, R. M., Geimer, S., Kröckel, U., Leiherer, A., Haller, K., Nemcová, Y. and Mages W. (2002) J. Cell Sci. 116 (8), 1449-1462.

contactauthor:RODRíGUEZ-RODRíGUEZ, M.

239


poster 131

Modulation of the activity of different variants of Bone Morphogenetic protein-2.Romero-Romero ML1, López-Lacomba JL2, Sanchez-Ruiz JM1

1Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, Granada, Spain.2Instituto de Estudios Biofuncionales, Universidad Complutense, Paseo Juan XXIII 1, 28040 Madrid, Spain

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily that induces bone formation and regeneration, and determines important steps during early stages of embryonic development.

Based on their ability to initiate ectopic bone formation, BMP-2 is used as therapeutic agent for bone repair, where enhancing bone regeneration through increasing BMP signaling has become standard practice in spine fusion surgeries and tibiae fracture healing.

Using a simple evolutionary hypothesis we chose different BMP2 variant trying to increasing the protein activity. In order to see if these single mutations affect in their biological activity, the variants were tested for their capabilities to induce alkaline phosphatase (ALP) expression in the myoblast cell line C2C12.

Pasted Graphic.pict ¬

Most of BMP-2 variants show a considerable variability in the capacity to induce ossteogenic differentiation. The analysis of the set of all variants revealed that the mutations selected have a great capacity for modulation of BMP activity.

contactauthor:roMero-roMero,Ml

240

poster 132

role of charge neutralization in the folding of the carboxy-terminal domain of histone h1.Alicia Roque, Núria Teruel, Inma Ponte, Pedro Suau.

Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain.

H1 linker histones are involved in chromatin structure and gene regulation. The carboxy-terminal domain (CTD) of histone H1 is very basic with approximately 40% Lys residues, approximately 75% of which are present as doublets. The CTD has little structure in diluted solution but becomes cooperatively folded upon interaction with DNA. The DNA-bound CTD contains alpha-helix, beta-structure, turns, and flexible regions. We studied the effects of char-ge neutralization on the secondary structure of the CTD independently of DNA interaction through deprotonation of the epsilon-amino groups of the Lys side chains at alkaline pH. Alkaline pH induces extensive folding of the CTD with proportions of secondary structure similar to those observed in the complexes with DNA. The CTD is phosphorylated by cyclin-dependent kinases. In the fully phosphorylated CTD, alkaline pH induces a higher amount of beta-sheet and a lower amount of alpha-helix, as observed in the complexes with DNA. These results, together with structure predictions, suggest that the increased hydrophobicity of Lys side chains accompanying charge neutralization is responsible for the folding of the CTD upon interaction with DNA.

contactauthor:Alicia ROQUE

241


poster 133

analysis in response to osmotic and heat stress in ustilago maydis.Karina Salmerón-Santiago1, Juan Pablo Pardo-Vázquez2, Oscar Flores-Herrera2 and Guadalu-pe Guerra-Sánchez1*1,1*Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, IPN. Prolong. de Carpio y Plan de Ayala S/N. C.P 11340. México D.F. 2Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria. C.P 04510, México, D.F.

Ustilago maydis is the agent responsible for corn smut. The ability of U. maydis to adapt to high environmental osmolarity and exposition to higher temperatures are process that sub-sequent loss viability [1]. Trehalose is a disaccharide which serves as a carbohydrate reserve and stress protector [2]. We investigated the changes in the concentration of trehalose, glyce-rol and trehalase activity in the response of U. maydis FB2 yeasts to thermal, sorbitol and NaCl stresses. When the cells were grown with 1M NaCl, 1M D-sorbitol or 37°C, cells grown at low rate than the control and at 40°C cells stop growing. The results showed that only sorbitol stress induced a sustained high trehalose accumulation while a transient accumulation occur under NaCl and heat stress The trehalase activity in cell extracts of 1M sodium chloride did not show increased activity, however it was 2-fold higher with 1M D-Sorbitol compared with sodium chloride or 37°C stress. This results suggests that trehalose pathway in U. maydis differs from those reported in bacteria and plants. U. maydis has to face the internal osmotic media of maize plant. It may be the reason why osmotic stress seems to be more important to this fungal [3], where it has been demonstrated high levels of sorbitol dehydrogenase activity [4]. We found that during osmotic stress, cells did not accumulate glycerol. When the cells were observed using optical microscopy under these conditions cells has no effect on their morphology; in contrast under electron microscopy with sodium chloride the cells displayed normal morphologies, with less mitochondria and varying size that control cells. The cells exposed to sorbitol were empty and small, there was loose of membranous and vesicular sys-tem, the cell membrane is breaking in someone cells and the cell-wall thickness varies from one part of the cell to another. The amount of intracellular sodium under treatment with NaCl does not toxic for the cell. Our date indicate that U. maydis have to response by a different pathway to balance their osmotic pressure.

[1] Herrera, J., León, C., Guevara,O.L., Cárabez, T.A. (1995) Microbiology. 141, 695-703.[2] Yancey, P.H. (2005) J. Exp. Biol. 208, 2819-2830.[3] Namiko, S.N., Nobuhiro, N., Simon, M., Hajime, S., David J. (2006) Nature. 441, 227-230.[4] Ruiz, H. (2005) Eukaryotic cell. 4, 999-1008.

contactauthor:Karina SALMERóN-SANTIAGO

242

poster 134

involvement of ferredoxin-naDp+ reductase FaD and naDp+/h binding domains for optimal catalytic complex formation and hydride transfer efficiency.Ana Sánchez-Azqueta1, Matías Musumeci2, Eduardo Ceccarelli2 and Milagros Medina1

1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Insti-tute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, Spain.2Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), Facul-tad de Ciencias Bioquímicas y farmacéuticas, Universidad Nacional de Rosario, Argentina.

In photosynthetic organisms, the enzyme responsible for NADPH production is ferredoxin-NADP+ reductase (FNR). This FAD-containing reductase takes two electrons from two reduced ferredoxin (Fd) molecules, and transfers them to NADP+ in a single hydride transfer step. This later process has been reported to occur through the formation of two intermediate charge transfer complexes species: FNRox-NADPH and FNRred-NADP+ [1]. Several FNR regions have been proposed as crucial for optimal FAD and NADP+ orientation during the hydride transfer event. Thus, Y79, S59 and the 102-110 loop (Anabaena FNR numbering) of the FAD binding region, are highly conserved among plastidic FNRs and apparently must contribute to FAD allocation in the FAD-binding site. Therefore, these residues are expected to modulate the flavin oxido-reduction properties and might also contribute to the hydride transfer efficiency. In the NADP+ binding module, recent structural studies suggest that while E267 and E268 might contribute to the displacement of the aromatic terminal tyrosine during the catalytic event, the volume of residue C261 (C266 of pea FNR) is critical for the formation of a com-petent catalytic complex [2]. In order to establish the role of all these residues in the hydride transfer event we have produced either Anabaena or pea FNR site-directed mutants at these positions. The oxido-reduction properties of these variants, as well as their efficiency in for-ming the intermediate charge transfer species and in hydride transfer during the reaction with the coenzyme are here reported.

[1] Tejero J, Peregrina JR, Martínez-Júlvez M, Gutiérrez A, Gómez-Moreno C, Scrutton NS, Medina M (2007) Arch. Biochem. Biophys. 459(1), 79-90.[2] Musumeci MA, Arakaki AK, Rial DV, Catalano-Dupuy DL, Ceccarelli EA (2008) FEBS J. 275(6), 1350-66.

contactauthor:Ana SÁNCHEZ-AZQUETA

243


poster 135

interaction of nucleoside derivatives with the human concentrative nucleoside transporters hCnt1, hCnt2 and hCnt3 using electrophysiological techniques.1Carlos Sancho-Mateo, 1Edurne Gorraitz, 2Carmen Sanmartín, 3Itziar Pinilla-Macua, 3Marçal Pastor-Anglada, 1Mª Pilar Lostao1Dept. of Nutrition, Food Science, Physiology and Toxicology. University of Navarra, Pamplo-na, Spain,2Dept. Organic and Pharmaceutical Chemistry, University of Navarra, Pamplona, Spain. 3Dept. Biochemistry and Molecular Biology, University of Barcelona, CIBER EHD, Barcelona, Spain

Concentrative nucleoside transporters are membrane proteins involved in anticancer and antivi-ral nucleoside-derived drugs uptake. CNT1 transports pyrimidine nucleosides and is inhibited by adenosine. CNT2 transports purine nucleosides and the pyrimidine uridine, and CNT3 transports both pyrimidine and purine nucleosides [1]. In order to explore the structural requirement of the substrates of these transporters, in the present work we have synthesized uridine and adenosine derivates and investigated their interaction with the three human isoforms of the CNT family, using the two-ectrode voltage clamp technique applied to Xenopus laevis oocytes expressing these trans-porters [2].

The nucleoside derivates were synthesized by functionalizing uridine and adenosine with aromatic systems that were substituted in position 4´ with different groups, to control size and polarity, and bound by aliphatic chains of variable length in position 5 of the pyrimidinic base and position 6 of the purine base.

The uridine derivates were transported by hCNT1 with similar affinity than uridine (K0,5˜37 µM) and a maximum transport rate (Imax) that was 70-50% of that for uridine. At 1 mM concentration, these derivatives induced in hCNT2 only 15-30 % of uridine maximal current. Interestingly rat CNT2 showed Imax of 65-85 % of uridine Imax and similar K0,5 than uridine. In hCNT3 they were transported with higher affinity than uridine (K0,5˜3-8 µM vs. 10 µM) and a Imax of 80-60 % with respect uridine Imax. The adenosine derivates were inhibitors of hCNT1 with a Ki˜2-5 times higher than that for adenosine (6 µM). However, they were transported by hCNT3 with ˜30 times reduction in the affinity compared with adenosine (K0,5˜600 vs. 18µM) and half of it’s maximal transport rate.

In summary, both hCNT1 and hCNT3 can transport bulky derivates of uridine and adenosine (hCNT3) with similar affinities and maximum transport rates. For hCNT3 modifications in uridine are better tolerated than in adenosine. However hCNT2 seems to be more restrictive with respect to the size of the substituents even in comparison with its orthologous in rat.

This work was supported by Fundación Marcelino Botín and Ministerio de Ciencia y Tecnología (Spanish Government) Grants BFI 2003-01371 and SAF2008-00577. The groups are members of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF) (Grant BFU2007-30688-E/BFI).

contactauthor:Carlos SANCHO-MATEO

244

poster 136

atomic force microscopy-based molecular recognition of fibrinogen receptor on human erythrocytesFilomena A. Carvalho1, Simon Connell2, Gabriel Miltenberger-Miltenyi1,3, Sónia Vale Pereira3, Alice Tavares4, Robert A.S. Ariëns5, Nuno C. Santos1

1Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal.2School of Physics and Astronomy, University of Leeds, LS2 9JT Leeds, United Kingdom.3GenoMed Diagnosticos de Medicina Molecular, 1649-028 Lisboa, Portugal.4Serviço de Imuno-Hemoterapia, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, 1649-028 Lisboa, Portugal.5Division of Cardiovascular and Diabetes Research, Leeds Institute for Genetics, Health and Therapeutics, University of Leeds, LS2 9JT Leeds, United Kingdom.

The established hypothesis for the mechanism of erythrocyte hyperaggregation, a vascular risk factor, is due to an increase in plasma adhesion proteins, particularly fibrinogen. Fibri-nogen-induced erythrocyte aggregation was considered to be caused by non-specific protein binding to erythrocytes membranes. In contrast, platelets are known to have a fibrinogen inte-grin receptor expressed on the membrane surface (membrane glycoprotein complex αIIbβ3). We demonstrate, by force spectroscopy measurements on an AFM, the existence of a single molecule interaction between fibrinogen and an unknown receptor on the erythrocyte mem-brane, with a lower but comparable affinity relative to platelet binding (fibrinogen-erythrocy-te and –platelet average (un)binding forces were 79 and 97 pN, respectively). This receptor is not as influenced by calcium and eptifibatide (an αIIbβ3 inhibitor) as the platelet receptor. However, its inhibition by eptifibatide indicates that it is a αIIbβ3-related integrin. Results ob-tained for a Glanzmann thrombastenia (a rare hereditary bleeding disease caused by αIIbβ3 deficiency) patient show impaired fibrinogen-erythrocyte binding. Correlation with genetic sequencing data demonstrates that one of the units of the fibrinogen receptor on erythrocytes is a product of the expression of the β3 gene, found to be mutated in this patient.

contactauthor:Nuno C. SANTOS

245


poster 137

Spreading of persistent infections in heterogeneous populationsJoaquín Sanz1, Luis Mario Floría1,2, Yamir Moreno1,3.

1Institute for Biocomputation and Physics of complex systems BIFI.2Departamento de Física de la materia condensada, Universidad de Zaragoza.3Departamento de Física teórica, Universidad de Zaragoza.

Up to now, the effects of having heterogeneous networks of contacts have been studied mostly for diseases which are not persistent in time, i.e., for diseases where the infectious period can be considered very small compared to the lifetime of an individual. Moreover, all these previous results have been obtained for closed populations, where the number of individuals does not change during the whole duration of the epidemics. Here, we go one step further and analyze, both analytically and numerically, a radically different kind of diseases: those that are persistent and can last for an individual’s lifetime. To be more specific, we particularize to the case of Tuberculosis’ (TB) infection dynamics, where the infection remains latent for a period of time before showing up and spreading to other individuals. We introduce an epidemiolo-gical model for TB-like persistent infections taking into account the heterogeneity inherent to the population structure. This sort of dynamics introduces new analytical and numerical challenges that we are able to sort out. Our results show that also for persistent diseases the epidemic threshold depends on the ratio of the first two moments of the degree distribution so that it goes to zero in a class of scale-free networks when the system approaches the ther-modynamic limit.

contactauthor:Joaquín SANZ

246

poster 138

Function of the trwB transmembrane domain in the protein-protein interaction and cellular locationRosa .L. Segura1, Sandra Águila1, Ana J. Vecino1, Begoña Ugarte1, Fernando de la Cruz2, Félix M. Goñi1, Itziar Alkorta1

1Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Uni-versidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.2Departamento de Biología Molecular, Universidad de Cantabria (UC) e Instituto de Biomedi-cina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), 39011 Santander, Spain.

Bacterial conjugation is a process that implies the transfer of genetic material from one donor cell to a recipient one through a direct cell to cell contact [1]. The genetic information recei-ved is usually beneficial to the recipient cell, allowing a fast adaptation to the environment. These benefits will include the spread of antibiotic resistance among bacteria and due to the efficiency of the conjugative process, bacterial conjugation constitute could help to develop possible genetic tools to introduce genes in a controlate and directed manner.

Different proteins codified by the conjugative plasmid are necessary for the process to ha-ppen. They are classified into three groups: i) Dtr proteins, that participate in the ralaxosome formation, ii) Mpf proteins that take part in the pilus assembly, and iii) the coupling protein; which is an integral membrane protein linking the relaxosome to the DNA transport appara-tus [2].

Previous studies in our group indicate that the transmembrane domain (TMD) of TrwB most likely has a role beyond the mere anchoring of the protein to the cell membrane [3,4)]. TMD could interact with other conjugative proteins to detect signals that could activate the con-jugation process. One of these putative candidates could be TrwE, a conjugative protein of R388 plasmid [5].

The purpose of this work is to elucidate the domains involved in the interaction TrwB-TrwE. In order to so, we designed different deletion mutants and the interactions were analysed using the bacterial two hybrid system.

Also we study the cell location of TrwB and two other mutants, a soluble mutant and a trans-membrane domain mutant.

[1].- Thomas,C.M., and Nielsen,K.M. (2005) Nat Rev Microbiol 3: 711-721.[2].-Zechner,E.L., de la Cruz,F., Eisenbrandt,R., Grahn,A.M., Koraimann,G., Lanka,E. Muth,G., Pansegrau,W., Thomas,C.M., Wilkins,B.M. and Zatyka,M. (2000) Ed. Thomas, C. M. 88-173.[3].- Hormaeche,I., Iloro,I., Arrondo,J.L., Goni,F.M., de la Cruz,F., and Alkorta,I. (2004). J Biol Chem 279: 10955-10961.[4].-Hormaeche,I., Segura,R.L., Vecino,A.J., Goni,F.M., de la Cruz,F., and Alkorta,I. (2006). FEBS Lett 580: 3075-3082.[5].-Llosa,M., Zunzunegui,S., and de la Cruz,F. (2003) Proc Natl Acad Sci U S A 100: 10465-10470.

contactauthor:Rosa .L. SEGURA

247


poster 139

nMr structural studies on peptides mimicking the pre-membrane stem region of the hiV-1 gp41 proteinSoraya Serrano1, Nerea Huarte2, Beatriz G. de la Torre3, David Andreu3, José L. Nieva2, M. Angeles Jiménez1.1Instituto de Química Física Rocasolano. CSIC. Madrid, Spain. 2Biophysics Unit (CSIC-UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country, Bilbao. Spain.3Universitat Pompeu-Fabra, Barcelona, Spain.

The gp120/41 glycoprotein of HIV-1 promotes fusion between the viral envelope and the human plasma membrane of the CD4 target cell [1-2]. Merging of the lipid bilayers promoted by the trans-membrane gp41 subunit is dependent on two conserved hydrophobic ectodo-main sequences: the amino-terminal fusion peptide (FP) and the highly aromatic pre-trans-membrane region (preTM). Although the atomic structure of the prefusion gp41 ectodomain remains unknown, the hydrophobic FP and the preTM sequence were shown to be in contact contributing to maintain the prefusion structure of the gp41 stem region [2]. CD and IR stu-dies on two hybrid peptides: HybK3, encompassing the FP and preTM sequences connected through a flexible linker, and ScrK3, with the conserved core of FP scrambled, indicated that specific structural features, such as the formation of intermediate folded structure enriched in β-turns and α-helix at moderate low polarity of the medium, depend on the FP sequence [3]. To get deeper insights into these structures, peptides HybK3 and ScrHybK3 are being studied by NMR under different solvent conditions, i.e. mixed water/hexafluoroisopropanol and dodecilphosphocoline micelles.

[1] Lorizate M, Gómara MJ, de la Torre BG, Andreu D, Arrondo JLR, Nieva JL. (2006) J. Mol. Biol. 360, 45-55.[2] Lorizate M, de la Arada I, Huarte N, Sánchez-Martínez S, de la Torre BG, Andreu D, Arrondo JLR, Nieva JL. (2006) Biochemistry 45, 14337-14346.[3] de la Arada I, Julien J-P, de la Torre BG, Huarte N, Andreu D, Pai EF, Arrondo JLR, Nieva JL. (2009) J. Phys. Chem. B 113, 13626-13637.

contactauthor:Soraya SERRANO

248

poster 140

Characterization of the putative residues involved in the synthesis of FMn and FaD in FaD synthetase from Corynebacterium ammoniagenesAna Serrano1,2, Adrián Velázquez-Campoy1,2 and Milagros Medina1,2. 1Department of Biochemistry and Molecular and Cellular Biology. University of Zaragoza. Spain.2Institute for Biocomputation and Physics of Complex Systems. University of Zaragoza. Spain.

FAD synthetase (Flavin adenine dinucleotide synthetase) from Corynebacterium ammoniage-nes (CaFADS) is a bifunctional enzyme that catalyzes two sequential steps in the biosynthesis of FAD [1]:

fosforilation of RB to produce FMN: RB + ATP → FMN + ADP

adenylylation of FMN to produce FAD: FMN + ATP → FAD + PPi

The CaFADS structure presents two catalytic modules, a C-terminal with ATP:riboflavinkinase activity and a N-terminal with ATP:FMN adenylyltransferase activity [2]. Models for the inte-raction of the enzyme with the reaction substrates and products have been produced, allowing location of all the protein substrates in their putative binding pockets [3].

In the present work several mutants selected as potential residues involved in the interac-tion with the ligands and in the catalytic activity have been characterized. Mutants at H28, H31, R161, S164 and T165 in the N-terminal domain, and at T208, N210 and E268 in the C-terminal domain positions, have been produced. The interaction of FADS WT and the di-fferent mutants with their substrates and products (RB, FMN FAD, ATP and ADP) has been characterized by Isothermal Titration Calorimetry. Moreover, the riboflavinkinase and aden-ylyltransferase activities have been analyzed by HPLC in order to settle the critical residues involved in catalysis.

[1] Manstein, D.J. and Pai, E.F. (1986) J. Biol. Chem. 261, 16169-16173.[2] Herquedas, B., Martínez-Júlvez, M., Frago, S., Medina, M. and Hermoso, J.A. J. Mol. Biol. In press.[3] Frago, S., Martínez-Julvez, M., Serrano, A. and Medina. M. (2008) BMC Mycrobiology. 8:160.

contactauthor:Ana SERRANO

249


poster 141

new biophysical approaches using in the environmental situation Shamiyan A.G.

State Enginering University of Armenia, Department of Chemical technology and environ-mental engineering 105 Teryan Str., 0009 Yerevan Armenia

At present the problem of anthropogenic pollution is very serious. The intensive use of natu-ral resources in the mining and metallurgical industries in Armenia is the increasing content of most chemical elements, including heavy metals in soils, waters and plants. In order to evaluate these industries on the environmental situation, in particular on a vegetative cover, we have carried out this research.From the four various concerning polluted plots have been collected samples of the plant to determine the content of heavy metals in the aboveground part of the samples. Samples were crushed to the size of 5 ± 0,5 mm by homogenizer marks HM - 2300, with the rotation speed 1200 min-1. Further the crushed samples have been sub-jected to dehumidification at Т = 30±20С and relative humidity of ~1 %. Dehumidification kinetics was investigated by gravimetric method. Mineralization of plant samples was carried out by dry ignition methods in a muffle furnace at temperature 4500C and dissolved in 0,5 M HCl. The concentrations of metals in HCl solutions were determined by the method of AAS with a graphite furnace and pulverize GFA - EX7I. The received curve of dehumidifications kinetics was divided into two sections:

- The first fragment – the initial stage of dehumidification characterized by “fast” diffu-sion, described by the equation (1)

A = 6(D/π∙a2)1/2∙ τ1/2 (1)

- The second fragment – a final stage of process of the dehumidification, characterized by the “slow” diffusion described by the equation (2)

lnA = ln(6/π)2 – (D∙π2∙τ)/a2 (2)

In the above equations:

A = (m0-mτ)/(m0-m∞) - non-dimensional index of moisture; m0, mτ, m ∞ - respectively initial, ongoing and final masse of the sample, gr.; τ - duration of dehumidification, sec.; a - the defining linear geometrical size, m; D –coefficient of Fik’s diffusion, m2/sek ..

Comparison of diffusion’s parameters indicates good agreement between the values D/a2 for the four samples in both fragments of a kinetic curve. At the same time, it is easy to see that the correlation between the values of the parameters of diffusion and ion content of heavy metals observed only in the case of molybdenum ions. As the transition metal molybdenum is involved in the reactions of peroxidation of lipid structures of cellular membranes, which apparently is the cause of the influence of molybdenum content on the permeability of cellu-lar membranes wormwood. Results of this work may be used for environmental monitoring of pollution of heavy metal ions.

contactauthor:SHAMIYAN A.G.

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poster 142

Cardiac dynamics : Spiral drift and meandering in heterogeneous heart tissuesJ. Bragard1 and A. Simic1

1Department of Physics and Applied Mathematics, University of Navarra, Pamplona, Spain

Cardiovascular diseases remain the major death cause in the industrialized countries. Nume-rical modeling of the electrical heart activity has proven to be very helpful in testing the different theories of arrhythmia genesis. Nowadays there exists a large variety of detailed numerical models describing closely the dynamics of the electric waves that propagate inside the heart tissue. In the present study, we have tested two such models, i.e., the Luo-Rudy I or Luo-Rudy passive model ([1]) and Luo-Rudy II (further extended to the LR dynamic model) ([2]). As heterogeneities are very important in the genesis and stability of cardiac arrhythmias, we have numerically investigated the drift of an induced spiral in a piece of two dimensional heterogeneous tissue for the two above mentioned models. The results show the large dispa-rity of drift between the two models.

[1] C. Luo, Y. Rudy (1991), Circ Res 68 : 1501-1526[2] C. Luo, Y. Rudy (1994), Circ Res 74 : 1071-1096[3] K. H. W. J. Ten Tusscher, A. V. Panfilov (2003), Am J Physiolo Heart Circ Physiol 284 : H542- H548

contactauthor:A. SIMIC

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poster 143

Modifying the flexibility of bacteriorhodopsin helices impairs proton transport efficiencyRosana Simón-Vázquez1, Tzvetana Lazarova1, Alejandro Perálvarez-Marín2, José Luís Bourde-lande3, Esteve Padrós1

1Centre d’Estudis en Biofísica, and Unitat de Biofísica, Departament de Bioquímica i de Bio-logia Molecular. Facultat de Medicina. Universitat Autònoma de Barcelona. 08193 Bellaterra. Spain.2Brigham and Women’s Hospital, Francis St. 75, Boston, MA 02115 (USA).3Departament de Química. Facultat de Ciencies. Universitat Autònoma de Barcelona.

Membrane receptors and transport proteins undergo conformational changes during their biological function. To discern which conformational changes are essential for the function and to which extent, have remained a difficult issue in the characterization of their molecular mechanism.

With the aim to characterize critical conformational changes associated to bacteriorhodop-sin function we have used mutagenesis combined with biophysical techniques, allowing the study of the protein in close physiological conditions. The light-driven proton pump bacte-riorhodopsin (bR), a membrane protein which belongs to the 7TM superfamily, converts light energy into an electrochemical gradient by transporting protons from the cytoplasmic to the extracellular side, giving place to the bR photocycle [1]. To favour the proton uptake from the cytoplasmic side it is believed that helix F moves outwards (together with loop E-F) and helix G moves inwards the protein normal [2].

To characterize the functional relevance of those movements we have studied two bR mutants with modified helix flexibility. The first is the Loop5 mutant, a chimeric bR where the native loop E-F has been replaced by its homologous in Rhodopsin, thirteen aminoacids longer and with an increased flexibility. The second is the E166C/A228C mutant which consists of a double mutation to cysteine to allow the cross-linking of helices F and G, blocking their possi-ble movement during the photocycle. This cross-linking can be reverted by using a reducing reagent to enable the unequivocal assignment of the effects observed in the mutant to helix restricted flexibility and not to the double mutation.

Flash-photolysis and FTIR has been used to study photocycle intermediates. Proton pumping activity has been determined by the transient absorbance change of the pyranine dye after protein photoexcitation. Transport efficiency was determined as the change of pH in the me-dium of proteoliposomes suspensions under illumination.

Loop5 chimera showed an altered photocycle with a preferential opening to the cytoplasmic side reflected in a higher accumulation and longer lifetime of N intermediate. In E166C/A228C mutant the photocyle seems to stop at M intermediate (deprotonation of bR) and the photocyle follows an alternative pathway. Those effects were reverted when the disulfide bridge was disrupted [3].

Any of the mutants has no O intermediate formation (reprotonation of bR from the medium), or very little to be detected, and an altered transport efficiency being about half of the effi-ciency of wt bacteriorhodopsin.

252

[1] Goldschmidt, C. R., Ottolenghi, M., and Korenstein, R. (1976) Biophys J 16, 839-43.[2] Hirai, T., Subramaniam, S., and Lanyi, J. K. (2009) Curr Opin Struct Biol 19, 433-9.[3] Simon-Vazquez, R., Lazarova, T., Peralvarez-Marin, A., Bourdelande, J. L., and Padros, E. (2009) Angew Chem Int Ed Engl 48, 8523-5.

contactauthor:rosanasIMón-Vázquez

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poster 144

approaching Fluoquinolone Drug Delivery SystemsIsabel Sousa, Paula Gameiro

Requimte, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal

Quinolones are a very well know class of antibacterial agents, as well as one of the most pres-cribed drugs in medicine for treatment of various bacterial infections. This wide use seems to be the main cause for bacterial resistance and, with increasing menace of bacterial resistance, constant development of new drugs and strategies to increase their efficacy is of great impor-tance. Although highly prescribed, these antibacterial drugs are known for their various side effects and toxicity, and some of the agents have been withdrawn or not approved for use.

For the past few years, drug delivery systems have been the target of intense research due to their aim to achieve a greater efficacy in the site of action as well as to improve aspects such as pharmacokinetics and/or minimizing side effects, contributing to the development of these systems.

Liposomes are frequently used as drug delivery systems due to their high versatility and bio-compatibility and are considered for drug delivery when therapeutic agents are toxic, have high potency and low blood circulation times. Encapsulation of drugs, such as antifungal agents, has been reported and even commercialized, but research, regarding quinolones and liposomes, consists, mainly, in membrane permeability and physicochemical studies.

Lipid formulations for drug delivery of similar fluoroquinolones were prepared and studied. Synthesis of Fluoroquinolones ternary complexes has also been performed.

contactauthor:Isabel SOUSA

254

poster 145

effect of chitosan degradation on its interaction with beta-lactoglobulinHiléia K. S. Souza1,2, Maria do Pilar Gonçalves1 and Javier Gómez2

1REQUIMTE, Chemical Engineering Department, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.2IBMC - Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avda. de la Universidad s/n, 03002 - Elche – Spain.

The characterization of complex formation between proteins and polysaccharide-based polye-lectrolytes has been widely studied in the last years due to their relevance in many biological systems and their applications in the development of products for the pharmaceutical and food industries [1]. The formation of protein-polyelectrolyte complexes (PPC) arises from the establishment of favourable inter-molecular interactions, mainly electrostatic in origin but complemented with hydrogen bonding and hydrophobic interactions [2-3].

Isothermal Titration Calorimetry (ITC) and Optical Dispersion (O. D.) measurements have been used to monitor the formation of complexes between β-lactoglobulin (β-Lg) and chi-tosan as a function of average molecular weight of chitosan (submitted to ultrasonication for different periods of time: 0, 5, 15 and 30 min), protein charge (in the pH interval 3 – 6, below and above the isoelectric point of the protein) and the ionic strength of the solution.

Both ITC and O.D. measurements confirmed the formation of coacervates between β-Lg and chitosan as well as soluble complexes at pH > 5. The enthalpy of complex formation was exo-thermic and dependent upon the time of degradation of chitosan (average molecular weight), concentration ratio of the interacting species and ionic strength of the solution. As pH is reduced below the isoelectric point of the protein, a drastic decrease in the affinity of the protein for chitosan is observed, revealed both by an almost negligible enthalpy of interaction (at any protein to chitosan molar ratio and ionic strength. Parallel turbidity measurements, above and below the isoelectric point of the protein confirmed that the establishment of complementary electrostatic interactions is a key factor in the molecular recognition process between β-Lg and chitosan. Furthermore, the dependence of the coacervate formation pro-cess on the protein to chitosan concentration ratio was explained in terms of the effect that the concentration-dependent stoichiometry of the complex has on its own solubility leading to the re-dissolution of the coacervate as the chitosan concentration is increased.

The financial support from FCT to REQUIMTE and the postdoctoral fellowship to H.K.S.S (SFRH/BPD/37514/2007) are greatly acknowledged.

[1] Turgeon S.L., Schmitt C., Sanchez C. (2007) Current Opinion in Colloid & Interface Science 12,166–178.[2] Guzey D., McClements DJ.(2006) Food Hydrocolloids, 20,124–131.[3] Souza H.K.S., Bai G., Gonçalves M.P., Bastos M. (2009) Thermochimica Acta, 495,108-114.

contactauthor:Hiléia K. S. SOUZA

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poster 146

Function and regulation of Vitis vinifera aquaporins heterologously expressed on yeastLuís Leitao1, Ana P. Martins2, Ana Madeira2, Catarina Prista1, Maria C. Loureiro-Dias1, Teresa Moura2, Graça Soveral2,3

1Instituto Superior de Agronomia, UTL, 1349-017 Lisboa, Portugal2REQUIMTE, Dep. Quimica, FCT-UNL, 2829-516, Caparica, Portugal3Faculdade de Farmácia de Lisboa, 1649-003 Lisboa, Portugal

Aquaporins (AQP) are ubiquitary membrane protein channels with critical roles in controlling cell and tissue water fluxes. Due to their high isoform multiplicity in plants, elucidation of the physiological function of each isoform has been a difficult task. This can be overcome using yeast, which proved to be suitable for heterologous protein expression.

For a better understanding of Vitis vinifera cv. Touriga Nacional aquaporin function and stress defense responses, an important step is the characterization of these water transporters. We identified five putative aquaporin genes homologous to plasma intrinsic proteins (PIP2;2 and PIP1;1) and tonoplastic intrinsic protein (TIP2;1). Heterologous expression in a S. cerevisiae mutant lacking native aquaporins has been performed and chimeric GFP-aquaporin fusion proteins showed their localization in the plasma membrane. Aquaporin function was assessed in intact transformant yeast cells by imposing osmotic gradients of an impermeant solute and following the time course of water fluxes in a stopped-flow fluorescence device. Moreover, regulation of Vitis aquaporin by a gating mechanism involving its protonation under cytosolic acidification was also investigated.

The osmotic permeability coefficients (Pf) obtained for yeasts expressing individually PIP2;2 and PIP1;1 were similar to the deprived aquaporin strain (with high activation energy (Ea) of water transport), suggesting a non-significant water transport route through these channels under tested conditions. However, for TIP2;1, Pf increased up to 4.5 fold (lower Ea) when compared to control values. Our results also evidenced a intracellular pH dependent aquapo-rin regulation.

contactauthor:Graça SOVERAL

256

poster 147

Conjugated Linoleic acid impairs adipose plasma Membrane permeability and FluidityAna P. Martins1, Paula A. Lopes2, Susana V. Martins2, Ana Madeira1,3, Nuno C. Santos4, José A. M. Prates2, Teresa F. Moura1, Graça Soveral1,5

1REQUIMTE, Dep. Química, FCT-UNL, 2829-516 Caparica, Portugal2CIISA, Faculdade de Medicina Veterinária, TULisbon, 1300-477 Lisboa, Portugal3Institute for Research in Biomedicine (IRB, Barcelona), Spain4Instituto de Medicina Molecular, Faculdade de Medicina-UL, Lisboa, Portugal5Faculdade de Farmácia-UL, Lisboa, Portugal

The relationship between the dietary ingestion of conjugated linoleic acid (CLA), a fatty acid frequently used as a body fat reducing agent, and its presence on cell membranes possible affecting cellular functions is still unexploited.

In this study, obese Zucker rats were fed atherogenic diets containing distinct saturated fats of vegetable or animal origin (palm oil or ovine fat, respectively), with or without 1% of CLA. Plasma membrane vesicles obtained from visceral white adipose tissue were used to assess the effectiveness of dietary fat and CLA isomers membrane incorporation, and its outcome on permeability (stopped flow light scattering) and fluidity (DPH and TMA-DPH fluorescence probes) to water and glycerol. These membrane vesicle preparations were enriched in the adipocyte plasma membrane fraction, as proved by the contents of caveolin, GLUT4, and AQP7. Moreover, they have shown to be osmotically responsive and therefore suitable for transport experiments. The lipid profile analysis of adipose membranes has shown that CLA supplementation did not affect cholesterol and total fatty acids membrane incorporation. Yet, the deposition of cis9,trans11 CLA isomer was higher than the trans10,cis12 isomer. A sig-nificant CLA effect on both water and glycerol permeabilities was detected, having dietary groups with CLA lower permeability values. The high activation energy for both water and glycerol transport suggests that permeation occurs mainly through bilayer diffusion. A sig-nificant decrease in adipose membrane fluidity was correlated with changes in permeability, which seem to be caused by the incorporation of the trans10,cis12 CLA isomer. Altogether, these results suggest that CLA supplementation through its incorporation in membrane phos-pholipids has an effect on the fluidity of adipose membranes possibly due to the formation of raft-like micro-domains that promote a decrease in permeability.

contactauthor:Graça SOVERAL

257


poster 148

understanding lactose permease lipid selectivity from aFM observations of supported planar bilayersCarme Suárez-Germàa, Oscar Domènecha, Laura Picasa, Jordi Hernández-Borrella,b , M. Teresa Monteroa,b, aDepartament de Fisicoquímica, Facultat de Farmàcia UB, 08028-Barcelona.bInstitut de Nanociència i Nanotecnologia de la Universitat de Barcelona (IN2UB). 08028-Bar-celona, Spain

The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better in pre-vious works we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labe-led phospholipids (Pyr-PE and Pyr-PG) at 37 ºC in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE:PG proportion found in the E.coli inner membrane (3:1, mol/mol) [1]. The main conclusion raised from this work was that LacY resides preferentially into liquid-crystalline phases. These observations has been corroborated by atomic force microscopy (AFM) ob-servations carried out on supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glyce-ro-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), which display clear gel/liquid-crystalline phase separation under the conditions of our studies [2]. In order to better investigate the existence of laterally segregated phospho-lipid domains we have extended our AFM observation to SLBs formed by DOPE:POPG and DPPE:POPG.

[1] L. Picas, C. Suárez-Germà, M. T. Montero, J. L. Vázquez-Ibar, Jordi Hernández-Borrell, Manuel Prieto, Luís M.S.Loura Biochimica et Biophysica Acta doi:10.1016/j.bbamem.2010.05.012[2] L.Picas.L. A. Carretero-Genevrier, M. T. Montero, J.L. Vázquez-Ibar, B. Seantier, P.E. Milhiet, J. Hernán-dez-Borrell Biochimica et Biophysica Acta 1798 (2010) 1014–1019

contactauthor:Carme SUÁREZ-GERMÀ

258

poster 149

Studying of therapeutic activity of an artemisia absinthium `s extract at level of bilayer lipid membranesSukiasyan A.R., Hambardzumyan A.F., Tadevosyan A.V.

State Engineering University of Armenia, Department of Chemical Technology and Environ-mental Engineering, 105, Teryan Str., 0009 Yerevan, Armenia. [email protected]

It is know that most physiological activities involve some kind of lipid-based receptor-ligand contact interactions. In this work was investigated the influence of the plant extracts of Ar-temisia Absinthium on some electrical properties of bilayer lipid membranes (BLM) which was obtained from the total phospholipids of a cow brain. Now we presented the results in relationship between by the influence of the plant extracts of Artemisia Absinthium L. on some electrical properties of BLM, both products of lipid peroxidation (LPO) and peroxidase activity of extract. We obtained the dose and thermal depending effects for our samples also. All results were presented by relatively of the total protein of sample.

After forming a “black lipid film” in membrane washing liquid was added a plant extract. For our experiments the membrane washing liquid was 0.1M solutions of KCl, NaCl, CaCl2. Plant extract was prepared on base those solutions. The electrical properties of BLM evaluated by both a specific membrane resistance (gm= 10-9Ω cm-2) and a membrane breaking potential (Ubp=mV). After adding the plant extract in KCl, NaCl, CaCl2 membrane washing liquids the value of gm changed accordingly on two-four times. In during of those processes observed for value of Ubp both increase on 6-10% in mono-valent area (K+, Na+) and decrease on 5% in bi-valent area (Ca 2+). The presence of the extract of Artemisia Absinthium affects the elec-troporation of the BLM by changing its mechanical properties. Transport of ions such as K+, Na+, and Ca 2+ through BLM pores discharge the membrane potential and durations tends to cause a dielectric breakdown of the BLM in presence of that plant extract. These results were statistical processing with special computer program MatLab, and can be useful in clinical evaluation of actualities of drug plants by antioxidant therapy.

So, our obtained results are of current interest for biomedical research with using Artemisia Absinthium as anti-inflammatory drug. Those investigations were supported ANSEF grant 07-NS-biochem-1440

contactauthor:SUKIASYAN A.R.

259


poster 150

Structure of molten globule by equilibrium ф-analysis: helicobacter pylori apoFLaVoDoXin Renzo Torreblanca Gonzáles 1, 2; Javier Sancho 1, 2

1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universi-dad de Zaragoza. Pedro Cerbuna 12, Zaragoza 50009, España.2Instituto de Biocomputación y Física de Sistemas Complejos, Universidad de Zaragoza, Mariano Esquillor s/n - Edificio I + D, 50018 Zaragoza, España.

Flavodoxin (Fld) is an essential protein [4] of Helicobacter pylori (Hp), a gram-negative bac-teria specialized in human stomach infection. Because, current treatments involving antibio-tics [6] are increasingly causes of resistences [5] it is therefore necessary to develop new treatments. Fld is not present in humans, which makes it a promising candidate for target-oriented drug development for the erradication of Hp. On the hand, the investigation of partially folded conformations of proteins is important for understanding the folding and dymanics protein. Apoflavodoxin under certain conditions, adopts partially folded conforma-tions close the native state, which are never the predominant species in equilibrium, but may be involved in the folding and biological function of the protein. One of these intermediate is the ¨molten globule ¨(MG), which increase its population at pH 2.0 [2] and whose struc-ture is unknown. We have used Ф-analysis [3] which was initially developed to investigate transition state of protein folding and has been extended to determine low-resolution three-dimensional structures of equilibrium intermediates [1]. Using site-directed mutagenesis, we have designed tens of mutants in which interactions between secondary structural elements have been broken, and we have obtained and analyzed then. Measurement of conformational stability by urea denaturation at pH 7 and pH 2 allows us to determine Ф values to derive a low-resolution structure of the molten globule.

[1] Campos, L., M. Bueno, J. López-Llano, M. Jiménez & J. Sancho (2004). J Mol Biol, 344, 239-55.[2] Cremades, N., M. Bueno, J. Neira, A. Velázquez-Campoy & J. Sancho (2008). J Biol Chem, 283, 2883-95.[3] Fersht, A., A. Matouschek & L. Serrano (1992). J Mol Biol, 224, 771-82.[4] Freigang, J., K. Diederichs, K. Schäfer, W. Welte & R. Paul (2002) Protein Sci, 11, 253-61.[5] Raymond, J., D. Lamarque, N. Kalach, S. Chaussade & C. Burucoa (2010) Helicobacter, 15, 21-7.[6] Seppälä, K., T. Kosunen, L. Veijola, P. Sipponen, P. Arkkila, H. Rautelin & R. Tilvis (2008) Scand J Gastroenterol, 43, 1149-50.

contactauthor:Renzo TORREBLANCA GONZÁLES

260

poster 151

Synaptic vesicle expression of vaCht-phluorin in septal neuronsLaura Torres-Benito1, Rocío Ruiz1, M. Ángeles Montes1, Guillermo Álvarez de Toledo1, Lucía Tabares1.1Departamento de Fisiología Médica y Biofísica. Facultad de Medicina. Universidad de Sevilla.

Fusion of pHluorin (pH-sensible green fluorescent protein) with distinct synaptic vesicle pro-teins, such as synaptobrevin, synaptophysin or the vesicular glutamate transporter (vGLUT), allows real time monitoring of vesicle fusion and. Recently, it has been generated a new fusion protein formed by the acetylcholine vesicular transporter (vAChT) and pHluorin [1]. This pro-tein can be used to monitor neurotransmission in cholinergic neurons from the septal region.

Septal neurons in primary culture in day in vitro 12 (DIV12) from P0-P1 Wistar rats were transfected with the vAChT-pHluorin construction which includes CMV as promoter. The level of expression and the subcellular localization of the protein vAChT-pHluorin, were checked by immunofluorescence with confocal microscopy. VAChT-pHluorin protein, detected by anti-GFP antibody, showed a punctate appearance over the neurites and colocalize well with the synaptic vesicle protein SV2. Likewise, the degree of expression of vAChT-pHluorin in cho-linergic neurons (anti-vAChT), GABAergic (anti-GAD65) and glutamatergic (anti-vGLUT) was studied.

Functional imaging experiments in transfected neurons showed that both electrical stimula-tion and high potassium solution (70 mM) exposure produce an increase in fluorescence in synaptic boutons located over neurites and soma in synaptic vesicles. As well, the application of 50 mM NH4 solution to transfected neurons produces a higher increase in fluorescence than depolarization. This is because all the pHluorin is lighting by the basification of the in-tracellular organelles. These results demonstrate the functionality of this new construction and its possible interest for monitoring exo-endocytosis in real time in synaptic boutons of septal neurons.

[1] Brauchi, S., G. Krapivinsky, L. Krapivinsky, and D.E. Clapham. (2008) Proceedings of the National Academy of Sciences of the United States of America. 105:8304-8.

contactauthor:Laura TORRES-BENITO

261


poster 152

Sar of hybrids of the microtubule stabilizing natural products dictyostatin and discodermolide. investigations into the relationship between binding affinity and cellular cytotoxicity.Chiara Trigili1, Guy J. Naylor2, Nicola M. Gardner2, Ian Paterson2, J. Fernando Díaz1 and Isabel Barasoain11Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.2University Chemical Laboratory, University of Cambridge, Cambridge, United Kingdom.

Microtubule stabilizing agents (MSA) bind to assembled microtubules, impairing the dynamics of these key cytoskeletal polymers. In this way, treated tumor cells are blocked in G2/M phase of the cell cycle leading to death via apoptosis. Paclitaxel, was the first MSA to be discovered and is currently used in the treatment of several cancers. Over the past few years, a structurally diverse set of natural products have been identified which share the ability of the taxanes to stabilize polymeric microtubules. This class includes the marine sponge-derived polyketides discodermolide[1] and dictyostatin[2]. Although these drugs have distinct chemical structures, they share a similar bioactive structure, displaying a common pharmacophore [3].

While many tumors initially respond favorably to treatment with Paclitaxel, its effectiveness can be limited by toxicity issues and the build up of drug resistance over the course of treatment. One of the main causes is overexpression of the MDR-1 gene, which encodes for

the drug efflux pump P-glycoprotein (P-gp). However, discoder-molide and dictyostatin are worst substrates for p-glycoprotein due to their higher hydrophobicity. Therefore, it is plausible that drugs developed using the discodermolide and dictyostatin scaffolds might be more effective against tumor cells overexpres-sing P-gp.

A set of eight derivatives of dictyostatin (modified at positions 2, 3, 6, 9, 10, 11 and 16[4]) and four hybrids of discodermolide and dictyostatin[5], have been evaluated to determine the structural features required for the interaction of these compounds with the Paclitaxel site on β-tubulin. Removal of the methyl group of dic-tyostatin at position 6, hydrogenation of the (Z)-alkene between positions 2 and 3 and substitution of the hydroxyl at position 9 with a methoxy group resulted in significant enhancements of dictyostatin’s binding affinity for the site. Intriguingly, the com-pound modified at position 9 displayed significantly activity against cells overexpressing p-glycoprotein, maintaining potency in these Paclitaxel-resistant cell lines.

[1] S. P. Gunasekera et al., J. Org. Chem., 1990, 55(16), 4912-4915.[2] G. Pettit et al., J. Chem. Soc., Chem. Commun., 1994, 1111-1112.[3] A. Canales et al., Chemistry, 2008, 14, 7557-7569.[4] I. Paterson et al., Bioorganic & Medicinal Chemistry, 2009, 17(6), 2282-2289.

262

[5] I. Paterson et al., Chem. Commun., 2010, 46(2), 261-263.

contactauthor:Chiara TRIGILI

263


poster 153

hamiltonian replica-exchange methods for protein-structure refinement including experimentally determined distance restraintsDunja Urosev1, Roman Affentranger1, Xavier Daura1,2

1Universitat Autònoma de Barcelona, Barcelona, Spain2Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain

Availability of high resolution protein structures is often limited due to complexity of the crystallization process or in the case of NMR due to incomplete set of NOE-derived distances. Hence, there is a need for alternative methods that would allow obtaining the equivalents of high-resolution protein structures from incomplete or low-resolution data sets. One of the potential approaches to tackle this problem is refinement of low-resolution protein structures using replica-exchange MD simulations with imposition of experimental restraints, such as structure factors or NOE distances, as the varying parameter.

Specifically, a cofilin homology model was subjected to REMD simulations, performed with restraining backbone-hydrogen NOE distances (from NMR data for cofilin structure), hence mimicking the ‘low-resolution-data scenario’. A region of approximately 10 residues presen-ted different conformation in the cofilin homology model (coil) and the actual cofilin structure (α-helix). A clear conformational change from coil into α-helix has been observed in a large number of replicas while a similar change was observed only in a fraction of conventional MD-simulation runs with time-averaged distance restraints. Unrestrained MD simulations did not result in any conformational change in this region. These preliminary results supported a potential of REMD simulations for protein-structure refinement and permitted the testing of different parametric choices, such as soft-core parameters for non-bonding potential terms [1], as well as exploration of applicative extent of this method in terms of the size of protein segment expected to undergo a conformational change. Results will be discussed.

[1] Affentranger, R., Tavernelli, I., and Di Iorio, E.E. (2006) J. Chem. Theory Comput. 2(2), 217-228.

contactauthor:Dunja UROSEV

264

poster 154

Mechanical properties of β-cateninAlejandro Valbuena1, Andrés Manuel Vera1, Javier Oroz1, Margarita Menéndez2 & Mariano Carrión-Vázquez1

1Instituto Cajal, CSIC, CIBERNED & IMDEA Nanociencia, Avda. Doctor Arce 37, E-28002 Ma-drid, Spain2Instituto de Química-Física Rocasolano, CSIC & CIBERES, Serrano 119, E-28006 Madrid, Spaine-mail: [email protected]

β-catenin is an important protein implicated in several biological processes, like cell adhesion and gene expression. In cadherin adhesions, β-catenin could function as a scaffold element binding classical cadherins to cytosqueleton through β-catenin and EPLIN [1]. Cadherin ad-hesions are under tension [2] and this mechanical force is able to activate, via β-catenin, the expression of proteins in charge of increasing the strength of the adhesion and remodelling it [3]. Thus, β-catenin is an important component of its mechano-transduction system. Howe-ver, nothing is known about how β-catenin responds to mechanical forces. Here, we have used AFM-based single molecule force spectroscopy (SMFS) to stretch β-catenin axially (end-to-end). The core of this structure, formed by 12 ARM repeats [4], shows very low mechanical stability (~50 pN at 0.4 nm/ms) of its ARM repeats. Interestingly, the presence of the two termini of β-catenin in this structure results in the occurrence of new force events (~10%) with an increased mechanical stability (~140 pN at 0.4 nm/ms), suggesting that both termini could mechanically stabilize the β-catenin core. Molecular dynamics simulations of the core fairly reproduce the experimental results. When the cadherin cytoplasmic tail is present as a complex the force pattern observed suggests that the β-catenin core could be mechanically stabilized when it interacts with cadherin cytoplasmic tail by all their interacting regions [5]. Our results suggest that the response to force of the β-catenin core is not as canalized as found in other solenoid proteins [6, 7], although the presence of the cadherin cytoplasmic tail seems to mechanically stabilize and canalize the response. It remain to be explored the response of β-catenin complexed in the native mechanotransduction system.

[1] Abe, K. & Takeichi, M. (2008) Proc Natl Acad Sci U S A 105, 13-9.[2] Ganz, A., Lambert, M., Saez, A., Silberzan, P., Bugin, A., Mege, R. & Ladoux, B. (2006) Biol. Cell. 98, 721-30.[3] Braga, V. (2002) Curr. Opin. Cell. Biol. 14, 546-56.[4] Huber, A., Nelson, W. & Weis, W. (1997) Cell 90, 871-82.[5] Xing, Y., Takemaru, K., Liu, J., Berndt, J., Zheng, J., Moon, R. & Xu, W. (2008) Structure 16, 478-87.[6] Li L, Wetzel S, Plückthun A, Fernandez JM. (2006) Biophys. J. 90:L30-2.[7] Lee G, Abdi K, Jiang Y, Michaely P, Bennett V, Marszalek PE. (2006) Nature 440:246-9.

contactauthor:Alejandro VALBUENA

265


poster 155

Discrete and continuous hybrid petri nets to analyze the dynamic behaviour of the h2o2-detoxifying pathway in chloroplastsEdelmira Valero1, Hermenegilda Macià2, Mª Isabel González1 and Valentín Valero3

1Department of Physical Chemistry, Industrial Engineering School, University of Castilla-La Mancha, Albacete, Spain. 2Department of Mathematics, Computer Engineering School, University of Castilla-La Man-cha, Albacete, Spain.3Department of Computer Science, Computer Engineering School, University of Castilla-La Mancha, Albacete, Spain.

Petri net theory is a mathematical formalism, enabling a formal and clear representation of bioche-mical networks at different abstraction levels, as well as their structural analysis. In contrast to the concepts of elementary modes and extreme pathways, Petri nets additionally provide analysis tech-niques for the computation of static and dynamic network properties [1]. Other strong advantages are the visual representation and animation facilities, which support the intuitive comprehension of the network and provide useful communication platform between theoreticians and experimen-talists. In the literature, there are two goal categories of Petri net biological modeling: qualitative and quantitative analysis. In the present communication we have applied Petri Nets to the study of the dynamic behaviour (quantitative approach) and properties (qualitative approach) of the H2O2-detoxifying pathway in chloroplasts. This is a complex biochemical network comprising a number of photochemical, spontaneous and enzymatic steps with a great variety of responses against en-vironmental changes. Previously, we performed a traditional representation using ordinary diffe-rential equations (ODEs) to model this biological system [2]. We have now extended this study by establishing the corresponding Petri net model, which allows us to have a graphical view of the biological process, and more importantly, it allows us to modify and analyze the model in an easier way than the corresponding ODEs. Thus, from the structural qualitative study of the Petri net 3 P-invariants (mass-conserving) have been obtained, and 26 T-invariants (state-conserving subnet-works). An examination of these T-invariants revealed the robustness of the system (directly related to ascorbate peroxidase activity), the redundancy of the pathway (the parallelism between chemical and enzymatic pathways to eliminate H2O2 in chloroplasts) and the importance of NADPH as the shared node for two related pathways: the Calvin cycle and the pathway under study.

[1] Koch. I., Heiner, M. (2008) in Analysis of Biological Networks (Edited by B Junker, F Schreiber), pp. 139-179. Wiley & Sons Book Seires on Bioinformatics (Series Eds. Pan Y, Zomaya, AY), Chapter 7.[2] Valero, E., González-Sánchez, M.I., Macià, H. and García-Carmona, F. (2009) Plant Physiology 149, 1958-1969.

Acknowledgments

This work was supported by Projects PEII09-0232-7745, PAI08-0175-8618 and POII10-0235-8597 from the Consejería de Educación y Ciencia de la Junta de Comunidades de Cas-tilla-La Mancha (JCCM, Spain).

contactauthor:Edelmira VALERO

266

poster 156

reconstitution into liposomes enhances nucleotide binding affinity of trwB conjugative coupling proteinAna Julia Vecino1, Rosa de Lima Segura1, Begoña Ugarte-Uribe1, Sandra Águila1, Fernando de la Cruz2, Félix M. Goñi1, and Itziar Alkorta1

1Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Uni-versidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.2Departamento de Biología Molecular, Universidad de Cantabria (UC) e Instituto de Biomedi-cina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), 39011 Santander, Spain.

Conjugative systems contain an essential membrane protein that couples the relaxosome to the DNA transport apparatus called coupling protein (T4CP). TrwB is the T4CP of the conju-gative plasmid R388. This protein, purified in the presence of detergents, binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, a soluble mutant, TrwBΔN70, binds uniformly all tested nucleotides [1]. Re-constitution of membrane proteins into liposomes is a widespread approach to analyze their biological function. Since, we are interested in the characterization of the activity of TrwB, we developed a reconstituted system that could be used to study the function of this protein. In this work, TrwB has been succesfully reconstituted into liposomes being the protein prefe-rentially outside oriented. The functional analysis of TrwB proteoliposomes demonstrate that when the protein is inserted into the lipid bilayer TrwB is selective for ATP and the affinity is enhanced compared with the protein purified in detergent and with a soluble deletion mutant, TrwBΔN70 [1]. From these results we conclude that the reconstitution of TrwB is the suitable way to study the biological activity of this coupling protein and that the transmembrane do-main of TrwB could play a regulatory role in the biological activity of TrwB.

[1] Hormaeche, I., Segura, R.L., Vecino, A.J., Goni, F.M., de la Cruz, F., and Alkorta, I. (2006) FEBS Lett 580, 3075-3082.

contactauthor:anajuliaVecIno

267


poster 157

Conformational Landscape of hepatitis C nS3 proteaseOlga Abian1,2,3, Sonia Vega1, Jose Luis Neira1,4, Adrian Velazquez-Campoy1,5

1Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zarago-za, Zaragoza, Spain2Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), ISCIII, Spain3Aragon Health Sciences Institute (I+CS), Zaragoza, Spain4Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche (Alicante), Spain5Fundación ARAID, Diputación General de Aragón, Spain

The non-structural protein 3 (NS3) of the hepatitis C virus possesses proteolytic activity in its N-terminal domain. The isolated protease domain exhibits similar proteolytic properties to those of the full length protein. NS3 protease has been considered a pharmacological target for drug development against hepatitis C virus, because it is responsible for the posttransla-tional processing of the non-structural region of the viral polyprotein. However, no clinical drugs based on NS3 inhibitors are currently available. NS3 protease is an allosteric enzyme; its activity is modulated mainly by NS4A, an accessory viral protein, and zinc.

The thermodynamic characterization of NS3 protease conformational stability and its inte-ractions with substrate and effectors (NS4A and zinc), through a combination of calorimetric and spectroscopic techniques, suggests a fairly intricate conformational landscape for this so-mewhat small protein (20 kDa), with different coexisting conformational states in equilibrium controlled by heterotropic allosteric interactions. This complex conformational equilibrium behavior provides the molecular and energetic basis for its allosteric regulation and valuable information for drug development strategies.

contactauthor:adrianVelazquez-caMpoy


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List of Contributors in alphabetical order

275


author pages

Abad, Enrique 124

Abian, Olga 106,267

Acosta, Jorge 129

Affentranger, Roman 40,263

Aguado-Llera, David 192

Águila, Sandra 246,266

Aguilella, Vicente M. 20,219,233

Aguilella-Arzo, Marcel 20,107

Aguilera, Adriana M. de 57

Ahijado-Guzmán R. 108,208

Ahyayauch, Hasna 109

Alcalde, Ana Isabel 110

Alcaraz, Antonio 20,219,233,232

Alés, Eva 129

Alfonso, C. 108,208

Alkorta, Itziar 246,266

Almeida, Zaida L. de 151

Alonso, Alicia 109,111,180

Alonso, Noelia 112

Alonso, Pablo J. 74

Aloria, Kerman 234

Alsina, M. Asunción 128

Altmann, Karl-Heinz 205

Álvarez de Toledo, Guillermo 84,113,114,260

Álvarez-Lacalle, Enric 216

Alves, Ema 115

Alves, Filipa 21

Amigó, Núria 216

Amorós, Diego 87

Andreu, David 247

Andreu, José M. 144

Apellániz, Beatriz 58

Aprile, Francesco A. 137

Araujo, Aitziber 117,176

Arbués, Ainhoa 210

Arias-González, José Ricardo 174,177

276

author pages

Arias-Moreno, Xabier 204

Ariëns, Robert A.S. 244

Arizmendi, Jesús M 234

Arnedo, María 229,230

Arrais, Dalila 119

Arranz, Rocío 117,140,176,238

Arregi, Igor 186

Arrondo, José Luis R. 13,116

Arruebo, Pilar. 110

Ausió, Juan 234

Baltazar, Carla 120

Ballesta, Juan P. G. 81

Bañares-Hidalgo, Ángeles 121

Bañó, Manuel 122

Bañuelos, Sonia 186

Baptista, António M. 152,195,197

Barasoain, Isabel 144,223,261

Basañez, Gorka 181

Bastos, Margarida 41,209

Beloso, Ana 32

Bellido, Elena 207

Bennouna, Mohammed 109

Bernadó, Pau 11,231

Bertoncini, Carlos W. 137

Bettinelli, Paola 77

Betz, William J. 82,133

Bicho, Ana 86, 141,168

Bielanska, Joanna 65

Bilbao, P. 116

Bizarro, Cristiano 59

Bolivar, Aniuska 70

Botelho, Hugo M. 60

Botta, Maurizio 144

Bourdelande, José Luís 251

Bouzat, Sebastián 123

Bragado-Nilsson, Elisabeth 32

277


author pages

Bragard, Jean 61,250

Bravo Yuste, Santos 124

Bretaña, Laura 234

Brito, Rui M.M. 151,178

Bross, Peter 170

Bruix, Marta 4,159,185,238

Bruscolini, Pierpaolo 125

Buceta, Javier 126

Bueren-Calabuig, Juan A. 127

Busquets, M. Antònia 128

Bustamante, Carlos 5

Busto, Jon V. 111

Cabeza, José María 129

Cabré, Elisa J. 130

Calero, Carles 107

Calvo, Ana C. 131

Campos-Prieto, Luis Alberto 132

Canales, Angeles 223

Canela Xandri, Oriol 126

Cano, Raquel 82,133

Cantalapiedra, Inma R. 61

Canto, António do 194

Caño-Delgado, Ana I. 70

Carcer, Guillermo de 32

Carloni, Paolo 142

Carrascosa, José L. 22,57,76,140,174,175,177,211

Carrión-Vázquez, Mariano 42,156,165,217,264

Carrodeguas, José Alberto 191

Carvalho, Ana Luísa 160

Carvalho, Filomena A. 244

Casademunt, Jaume 62,126,216

Casañas, Juan José 133

Cascante, M. 220

Cases, R. 228

Castaño, Raquel 159

Castón, José R. 22

278

author pages

Castro, Marta 110

Catalina López, Laura 191

Catapano, Elisa R. 189

Ceccarelli, Eduardo 242

Cecconi, Ciro 5

Ciero, Milagros 230

Clarke, Richard 137

Cláudio, Martinho 115

Coelho, Pedro 83

Comes, Núria 65

Conchillo-Sole, Oscar 134

Confraria, Ana 70

Connell, Simon 244

Contreras, Jorge 27

Corbalán-García, S. 163

Coutinho, Ana 30,136

Cremades, Nunilo 137

Cruz, Antonio 138,218,219

Cruz, Fernando de la 246,266,

Cruz, Pedro 178

Cruzeiro, Leonor 23

Cuevas, Carmen 127

Chaboy Nalda, Jesús 142

Chalmeau, J. 39

Chen, Allen Y. 137

Chenal, Alexandre 85

Chory, Joanne 70

D’Abramo, Marco 231

Damas, João M. 139

Daudén, María I. 140

Daura, Xavier 40,134,263

Davi, Marilyne 85

David, Miren 65

Delaarada, I. 116

Dias, Fernando M. V. 160

Dias, Pedro 141

279


author pages

Díaz Moreno, Irene 142,158

Díaz Moreno, Sofía 142

Díaz Quintana , Antonio 142,153,158,158

Díaz, Fernando 157

Díaz, José Fernando 223

Diez-García, Fernando 63

Dilão, Rui 143

Dill, Jesse 5

Dillingham, Mark S. 162

Dobson, Christopher M. 6,137

Dols, Aurora 166

Domènech, Òscar 128,257

Domingos, Patricia 141

Dunne, Paul 137

Dziembowski, A 221

Eaton, Peter 188

Echaide, Mercedes 190

Echebarria, Blas 61

Echenique, Pablo 149

Edwards, Martin 166

Elías-Arnanz, Montserrat 71

Elowitz, Michael 26

Encinar, José A. 213

Enciso, Marta 145

English, Douglas 132

Escudero, Isabel 237

Espinosa Angarica, Vladimir 146

Esquembre, Rocío 64,201,236

Esteban, Daniel 166

Esteban-Martín, Santiago 148

Estrada, Jorge 149

Estrada, P. 121

Fàbregas, Norma 70

Falces, Jorge 186

Falo, Fernando 67,123,150

Fang, Wei-Shuo 144

280

author pages

Faraudo, Jordi 107

Faria, Tiago Q. 151

Fedorov, Alexander 130

Feijó, José Alberto 86,141

Felipe, Antonio 65

Fenwick, Robert B. 148

Fernández-Ballester, Gregorio 213

Fernández-Recio, Juan 231

Fernando Díaz, J. 144,205,261

Ferreira, Patricia 66

Ferrer-Montiel , Antonio 169,213

Feyen, Fabian 205

Fiasconaro, Alessandro 67

Filipe, Luís C. S. 152

Fillat , María F. 72

Finn, Ron 234

Flint, SJ 226

Flores-Herrera, Oscar 241

Floría, Luis Mario 245

Fonseca Moura, Teresa 75,86,256

Font, Josep 185

Fontes, Carlos M. G.A. 160

Formosa, Pau 70

Frago, Susana 182

Franke, Katharina J. 35

Fritz, Günter 60

Frutos Beltrán, Estrella 142,153

Fuerst, Oliver 187,154

Fumagalli, Laura 166

Gago, Federico 127

Galera-Prat, Albert 156,217

Galmarini, Carlos M. 127

Gallego, Cristina 157

Gamba, Alessio 77

Gameiro, Paula 188,253

281


author pages

Gameiro, Paulo 68

Garcés, J. L. 220

García de la Torre, José 87

García, Carolina 81

García, Jose Luis 80

García, Pedro 80,157

García, Santiago 110

García-Alvarez, Begoña 160

García-Barrios, Alberto 237

García-Carmona, Francisco 167

García-Carril, Ana M. 144

García-Giménez, Elena 232,233,20

García-Heredia, José M. 158

Garcia-Manyes, Sergi 25

García-Mayoral, María Flor 159

García-Moreno, Diana 71

Garcia-Ojalvo, Jordi 26

Garcia-Parajo, Maria F. 43

García-Sáez, Ana J. 44

Gardner, Nicola M. 261

Gascón, Sonia 237

Gavilanes., José G. 173

Geeves, Michael A. 161

Gewartowski, K 221

Gierusz, Leszek A. 161

Gil, David 184

Gil-Rodríguez, María Concepción 229,230

Giraldez, Teresa 27

Girona , Victòria 128

Gollnick, Benjamin 162

Gomes , Moreira 115

Gomes, Cláudio M. 170,60

Gómez Hernández, María 164

Gómez Roberto, Josué 22

Gómez, Javier 192,254,213

Gómez-Fernández, J.C. 163

282

author pages

Gómez-Moreno, Carlos 207,72

Gómez-Pinto, Irene 63

Gómez-Puertas , Paulino 200,206,229,230

Gómez-Sicilia, Àngel 165

Gomila, Gabriel 166

Gonçalo, Rui 115

Gonçalves, Patrícia 168

Gonzalez Velasco, J. 116

González, Ana 80

González, Andrés 72

González, Carlos 63,185

González, Mª Isabel 265

González-Barroso., M. Mar 37

González-Ros, José M. 236

González-Sánchez, María I. 167

Goñi, Félix M. 109,111,180,246,266

Gorraitz, Edurne 243

Gramse, Georg 166

Granell, Meritxell 154,187,193

Grasa, Laura 110

Gregorio-Teruel, Lucia 169

Gros, Belén 110

Guallar, Victor 28

Guerra-Sánchez, Guadalupe 241

Guijarro, Iñaki 85

Guillén, Natalia 237

H. Jesus, Catarina S. 178

Haavik, Jan 131

Hambardzumyan, A.F. 258

Hearing, P. 135

Henriques, Barbara J. 115,170

Herguedas, Beatriz 171,203

Hermoso, Juan Antonio 80,171

Hernández-Borrell, Jordi 257

Hérnandez-Rocamora, Víctor M. 172

Herrero-Galán, Elías 173,174

283


author pages

Hervás, Rubén 217

Holmgren, Miguel 27

Hormeño, Silvia 175

Huarte, Nerea 117,176,247

Huguet, Josep 59

Ibañes, Marta 70

Ibarra, Borja 175,177,211

Iceta, Ruth 110

Jankowiak., R. 228

Jemal, Imane 113,114

Jiménez- Rojo, Noemi 180

Jiménez, M. Angeles 71,185,238,247

Jiménez, Mercedes 179,208

Jiménez-Barbero, Jesús 223

Jiménez-Jiménez, Jesús 37

Karst, Johanna 85

Kelleher, Joanne K. 68

Klenerman., David 137

Konarev, Petr 186

Kunert, Renate 196

Lacadena, Javier 173

Ladant, Daniel 85

Lagartera, Laura 157

Lakhanpal, Amit 26

Landajuela, Ane 181

Landeta, Olatz 181

Lans, Isaias 182

Larriva, María 183

Lasso, Gorka 184

Latorre, Eva 110

Laurence-Martínez, Luis 165

Laurents, Douglas V. 63,185,238

Lázaro, José M. 177

Lazarova, Tzvetana 251

Leblanc, Gérard 154,187,193

LeBon, Lauren 26

284

author pages

Lectez, Benoit 186

Leitao, Luís 255

León, Xavier 193

Li, Wei 7

Lidón López, M. 20,232

Lillo, Pilar 81,224

Lima Segura, Rosa de 246,266

Lin, Yi-Bin 154,187

Lindenberg, Katja 124

Lino, Rita R. 36

Liu, Jianwei 132

Lopes, Paula A. 75,256

Lopes, Sílvia 188

López Cascales , J. J. 78

López, G. 121

López-Alonso, Jorge P. 63,185

López-Lacomba, JL. 239

López-Montero, Iván 76,189,219

López-Pérez, Miriam 192

Lopez-Rodriguez , Elena 190

López-Viñas, Eduardo 229,230

Lórenz-Fonfría, Víctor A. 154,187,193

Lostao, Anabel 72,207

Lostao, M. Pilar 73,243

Loura, Luís 30,130,194

Loureiro-Dias , Maria C. 255

Lousa, Diana 195

Luis, Fernando 207

Luján, M.A. 228

Luna, Arilla 118

Luque, Daniel 22

Llorca , Oscar 160,227

Llorente, P. 228

Ma, Cong 7

Maccari, Giorgio 144

Macià, Hermenegilda 265

285


author pages

Machuqueiro, Miguel 152,197

Madeira, Ana 255,75,256

Madeira, Catarina 30

Madurga, S. 220

Maeso, Rubén 196

Magalhães, P. R. 197

Malumbres, Marcos 32

Mallavia, Ricardo 201

Manuel, Miguel 198

Manyosa, Joan 215

Marabini, Roberto 22

Marijuán, Pedro 210

Marín, Arturo 199

Márquez, Ileana 76

Marqusee, Susan 5

Martel, Fátima 31

Martel, P. 45

Martín, Carlos 210

Martín-Benito, Jaime 140,234

Martínez Buey, Rubén 79,112,223

Martínez del Pozo, Álvaro 173

Martinez, Aurora 131

Martínez, Jesús I. 74

Martínez, Juan E. 204

Martínez-Cruz, Alfonso 213

Martínez-Gil, Luis 122

Martínez-Gómez, Ana-Isabel 192

Martínez-Júlvez, Marta 171,203

Martínez-Pérez, María José 207

Martínez-Ripoll, Martín 80

Martínez-Rodríguez, Sergio 192

Martínez-Tomé, Maria José 201

Martín-García, Fernando 200,206

Martins, Ana P. 75,255,256

Martins, Jorge 119,198

Martins, Patrícia A. T. 202

286

author pages

Martins, Susana V. 75,256

Martos, Ariadna 179,208

Mas, F. 220

Maspoch, Daniel 207

Matesanz, Ruth 144,205

Matheus, Nyurky 110

Maximov, Anton 84

Mazo, Juan José 67,150

McKinney, Jeffrey 131

Medina, Milagros 66,118,171,182,203,242,248

Melo, Ana 136

Menao, Sebastián 229

Mendieta, Jesús 200,206

Mendieta-Moreno, Jesús I. 200

Mendoza, Carmen 110

Menéndez, Margarita 157,226,264

Menéndez-Conejero, R. 135,226

Mesa, Pablo 32

Mesonero, José E. 110

Metallo, Christian 68

Miguel, Rocío de 207

Miltenberger-Miltenyi, Gabriel 244

Mingarro , Ismael 122

Minton, A. P. 108

Mir, Pablo 27

Miramar, Mª Dolores 66

Mirassou, Yasmina 71

Mobashery, Shahriar 80

Moleiro, Lara H. 76,219

Moniz, Cláudia V. S. 178

Monroy, Francisco 46,76,189,219

Montero, Francisco 77,199

Montero, M.Teresa 257

Monterroso, Begoña 208

Montes Fernández, Mª Ángeles 113

287


author pages

Montes, María-Angeles 114,260

Montoya, Guillermo 32,221

Morales, María 80

Morán, Federico 77

Moreno, Maria João 202,209

Moreno, Sonia 76

Moreno, Yamir 210,245

Moreno-Herrero, Fernando 162,175

Mori, Mattia 144

Morín, Jose A. 177,211

Moro, Fernando 212,227

Morozova-Roche, Ludmilla A. 60

Moura, Teresa 168,255

Muga, Arturo 212,227,234

Müller, Ulrich 217

Muñoz López, Francisco 142

Muñoz, Inés G. 32

Muñoz, Victor 47,132

Muraro , Daniele 143

Murillo, Divina 110

Musumeci, Matías 242

Narayanan Naganathan, Athi 125

Navajas, Daniel 33

Navarro, Aaron 213

Navarro, Jorge 210

Naylor, Guy J. 261

Neher, Margret 214

Neira, José L. 192,213,58,196,117,176,247

Neves, Cristina 188

Nieva, José Luis 122,267

Nir, Shlomo 58

Nollmann, Marcelo 34

Northcote, Peter T. 223

Nuno Dias, Pedro 86

Nuño, Juan Carlos 199

author pages

Oliveira Costa, Sara D. 78

Olmeda, Bárbara 138

Olmo, Nieves 173

Olsen, Rikke K. 170

Oncins, G. 235

Oñaderra, Mercedes 173

Orel, Mikhail 215

Orlandi , Javier G. 62,216

Oroz, Javier 156,165,217,264

Orozco, Modesto 12,231

Orte, Angel 137

Ortega Portero, Esther 79

Ortega, Álvaro 87

Orzáez, Mar 44

Osada, Jesús de la 237

Ospina, Olga L. 190,218

Ostapchuk, P. 135

Padmanabhan, S. 71

Padrós, Esteve 215,193,251,154,187

Pandurangan, Bharathi 83

Pardo-Cea, Miguel A. 63

Pardo-Vázquez, Juan Pablo 241

Park, AhYoung 32

Parra, Elisa 219

Pascual, Jose Ignacio 35

Pastor, I. 220

Pastor-Anglada, Marçal 243

Paterson, Ian 223,261

Pearson, David S. 161

Peleato, María Luisa 66,72

Pena, A. 221

Peñaranda, Angelina 61

Pera, Benet 223

Perales, Judit 227

Perálvarez-Marín, Alejandro 251

Pereda, Jose Mª de 79,112,164,224,

289


author pages

Pereira, Inês A. C. 36

Pérez, Alejandra 73

Pérez, Rubén 225

Pérez-Berná AJ 135,226

Pérez-Calvo, María Angeles 227

Pérez-Dorado, Inmaculada 80

Perez-Gil, Jesus 121,130,190,138,218,219

Pérez-Lara, A. 163

Pérez-Payá, Enrique 44

Pey, Angel L. 131

Picas, Laura 257

Picorel, Rafael 74,228

Pié, Angeles 230,229

Pié, Juan 229,230

Pilar Gonçalves, Maria do 254

Pineda, Oriol 223

Pinheiro, Teresa J.T. 48,161

Pinilla-Macua, Itziar 243

Pinto, Sandra N. 64

Plaza, Miguel A. 110

Pons, Carles 231

Ponte, Inma 240

Poraso, Rodolfo D. 78

Poveda, José A. 64,236

Prada-Gracia, D. 150

Prado., Adelina 234

Prat, Josefina 128

Prates, José A. M. 75,160,256

Prieto, Jesús 213

Prieto, Manuel 30,64,130,136

Prista, Catarina 255

Puisac, Beatriz 229,230

Pujol, Jonai 73

Queralt-Martín, María 20,107,232,233

Raja, Sebastian 81

290

author pages

RAKA, N. 116

Ramanathan, Ravishankar 132

Ramos, Feliciano J. 230,229

Ramos, Isbaal 234

Ramos, Mónica 229

Raugei, Simone 142

Razzak, Mina 223

Redondo-Morata, L. 235

Renart, María L. 236

Requejo-Isidro, José 111

Rey, Antonio 145,183

Reyes Mateo, C. 201,64,236

Reyes, Alejandro 73

Rial, Eduardo 37

Ribeiro, V. 45

Ribó, Marc 185

Ricardo Arias-González, J. 57,175,211

Rico, Manuel 159,238

Ries, Jonas 44

Ritort, Felix 59

Rivas, Germán 108,172,179,208

Rizo, Josep 7

Robinson, Carol V. 32

Rodríguez-Rodríguez, M. 238

Rodríguez-Yoldi, Mª Carmen 237

Rodríguez-Yoldi, Mª Jesús 237

Romão, Maria João 160

Romero-Romero, ML. 239

Roque, Alicia 240

Rosa, Miguel A. De la 24,142,153,158,

Ruiz, Rocío 82,133,214,260

Ruiz-Molina, Daniel 207

Sacristán, Miguel A. 81

Sagués, Francesc 126

Sáiz, José Luis 157

291


author pages

Salas, Margarita 177,211

Salmerón-Santiago, Karina 241

Salvador, Armindo 83

Salvatella, Xavier 148

Salzano, Maria 158

San Martín, C. 135,226

Sánchez, Juan Carlos 140

Sánchez-Azqueta, Ana 242

Sánchez-Martinez, Silvia 122

Sanchez-Ruiz, Jose M. 49,239

Sancho, Javier 146,191,204,259,149

Sancho-Mateo, Carlos 243

Sandal, Massimo 137

Sanles, Reyes 80

Sanmartín, Carmen 243

Santiveri, Clara M. 71

Santoro, Jorge 71,185

Santos, Ana Isabel 168

Santos, Nuno C. 75,244,256

Sanz, F. 235

Sanz, Joaquín 210,245

Savageau, Michael A. 83

Scherer, Tanja 131

Schürch, David 218

Schwille, Petra 44

Segovia, Margarita 84

Seibert, M. 228

Serna, Marina 32

Serrano, Ana 248

Serrano, Soraya 247

Sesé, Javier 207

Shamiyan, A.G. 249

Shank, Elizabeth 5

Shi, Jing-Zhe 144

Simic, A. 250

Simón-Vázquez, Rosana 251

292

author pages

Soares, Cláudio M. 139,195,120

Sonnenberg, Arnoud 164,79,224,112

Soriano, Jordi 216

Sot, Jesús 111

Sotomayor Pérez, Ana-Cristina 85

Sousa, Isabel 253

Souza, Hiléia K. S. 254

Soveral, Graça 75,255,256

Sprinzak, David 26

Stephanopoulos, Gregory 68

Striker, Waldemar 80

Su, Lijing 7

Suárez-Germà, Carme 257

Suau, Pedro 240

Südhof, Thomas C. 84

Sukiasyan A.R. 258

Susana, Sonia 118

Susin, Santos 66

Svergun, Dmitri I. 231,186

Tabares , Lucía 82,133,214,260

Tadevosyan, A.V. 258

Taeusch, H.William 190

Taneva, Stefka G. 186,212

Tapia-Rojo, R. 150

Tavares, Alice 244

Tavares, Bárbara 86

Teigen, Knut 131

Teixeira, Miguel 8

Teixeira, V. 45

Tejero, Hector 199

Teresa-Rodrigo, María Esperanza 229,230

Teruel, Núria 240

Thibault, C. 39

Thöny, Beat 131

Tong, Liang 184

293


author pages

Torre, Beatriz G. de la 247

Torreblanca Gonzáles, Renzo 259

Torres-Benito, Laura 260

Trepat, Xavier 38

Treviño M. 238

Trigili, Chiara 144,223,261

Ugarte, Begoña 246,266

Urbaneja, María A. 186

Urien, Héctor 112

Urosev, Dunja 263

Valbuena, Alejandro 156,165,217,264

Vale Pereira, Sónia 244,267

Valente, Pierluigi 169

Valenzuela, Carmen 65

Valero, Edelmira 167,265

Valero, Valentín 265

Valpuesta, José M. 57,117,176,211,177,32,174,175,221,227,234

Valle, Mikel 140,184

Vaz, Winchil L. C. 202

Vázquez, Manuel 57

Vázquez, Sara 77

Vecino, Ana Julia 246,266

Velazquez-Campoy, Adrián 118,209,178,267,202,204,106,248,212

Velez, Marisel 50,76

Venceslau, Sofia S. 36

Ventura, Salvador 191

Vera, Andrés Manuel 156,264

Vicente, Miguel 179

Vicente, Raquel 110

Vieu, C. 39

Viguera, Ana R. 180

Vilanova, Maria 185

Vilaseca, E. 220

Villalonga, Núria 65

Villanueva, Raquel 66

Viñuales, Carmen 237

294

author pages

Vollmer, Waldemar 80

Wagner, Amin 142

Wang, Xiang 132

Wesch, Diana 27

Winge, Ingeborg 131

Xavier, Daniela 77

Xiang, Song 184

Xu, Yibin 7

Yanamandra, Kiran 60

Yébenes, Hugo 32

Yeeles, Joseph T. P. 162

Ying, Ming 131

Yruela, Inmaculada 74

Yu, Linda P.C. 184

Zabala, Juan Carlos 159

Zhou, Min 32

Zorrilla, Silvia 208


List of Participants in alphabetical order

297


Lastname Firstname affiliation email

ABIAN Olga I+CS [email protected]

AGUILELLA Vicente Universitat Jaume I [email protected]

AGUILELLA-ARZO Marcel University Jaume I [email protected]

AHIJADO GUZMÁN Rubén CIB-CSIC [email protected]

AHYAYAUCH Hasna Unidad de biofisica (UPV) [email protected]

ALCALDE Ana Isabel University of Zaragoza [email protected]

ALONSO Alicia Universidad del Pais Vasco [email protected]

ALONSO Noelia Center for Cancer Research [email protected]

ALVAREZ DE TOLEDO Guillermo Universidad de sevilla [email protected]

ALVAREZ LACALLE Enrique UPC [email protected]

ALVES Ema ITQB [email protected]

ALVES Filipa Instituto Gulbenkian de Ciencia [email protected]

ANDRACA Nagore Unidad de Biofisica.Centro mixto CSIC-UPV

[email protected]

APELLANIZ Beatriz UNIDAD DE BIOFíSICA (CSIC-UPV/EHU)

[email protected]

ARAUJO Aitziber Universidad del Pais Vasco [email protected]

ARILLA LUNA Sonia Susana

UNIVERSIDAD DE ZARAGOZA [email protected]

ARRAIS Dalila IBB-CBME [email protected]

ASTIER Yann Astier Instituto de Technologia Quimica e Biologica, Universidade Nova de Lisboa

[email protected]

BALTAZAR Carla ITQB-UNL [email protected]

BAÑARES-HIDALGO Ángeles UCM [email protected]

BAÑó POLO Manuel Universidad Valencia [email protected]

BAÑUELOS Sonia University of the Basque Country [email protected]

BAQUEDANO Silvia Universidad de Zaragoza [email protected]

BASTOS Margarida Facultyof Sciences [email protected]

BENSENY Núria UAB [email protected]

BERNADó Pau IRB Barcelona [email protected]

BIELANSKA Joanna Universitat de Barcelona [email protected]

BIZARRO Cristiano Universidad de Barcelona [email protected]

BOIRA BONHORA Xavier LOT - Oriel Gmbh ¬Co [email protected]

BOTELHO Hugo ITQB/UNL [email protected]

BOUZAT Sebastián Universidad de Zaragoza [email protected]

BRAGARD Jean university navarra [email protected]

BRAVO YUSTE Santos Univ. Extremadura [email protected]

BRUIX Marta IQFR-CSIC [email protected]

298


BRUSCOLINI Pierpaolo Universidad de Zaragoza [email protected]

BUCETA Javier Parc Cientific de Barcelona [email protected]

BUEREN CALABUIG Juan Antonio

Universidad de Alcalá [email protected]

BUSQUETS Maria Antonia

University of Barcelona [email protected]

BUSTAMANTE Carlos U. California, Berkeley [email protected]

CABEZA José María University of Seville [email protected]

CALVO Ana University of Bergen [email protected]

CAMPOS PRIETO Luis Alberto CIB - CSIC [email protected]

CANO GARCíA Raquel University of Seville [email protected]

CARNEIRO Jorge I. Gulbenkian de Ciencia [email protected]

CARRASCOSA José L. CNB-CSIC [email protected]

CARRIóN-VÁZQUEZ Mariano Instituto Cajal/CSIC [email protected]

CASADEMUNT Jaume Universidad de Barcelona [email protected]

CASTóN José CSIC-CNB [email protected]

CERRADA Alejandro Complutense University of Madrid. Faculty of Biology

[email protected]

CLADERA Josep UAB [email protected]

CLEMENTE Isabel Universidad de Zaragoza [email protected]

CONCHILLO SOLE Oscar IBB UAB [email protected]

CORTIJO Miguel UCM [email protected]

COUTINHO Ana Insituto Superior Técnico [email protected]

CREMADES Nunilo University of Cambridge [email protected]

CRUZ RODRíGUEZ Antonio Universidad Complutense de Madrid [email protected]

CRUZEIRO Leonor University of Algarve [email protected]

CUELLAR Jorge NATIONAL CENTER OF BIOTECH-NOLOGY

[email protected]

CHACON Pablo CIB-CSIC [email protected]

DAMAS João M. ITQB-UNL [email protected]

DAUDéN María I. CNB-CSIC [email protected]

DAURA Xavier ICREA y UAB [email protected]

DE ALMEIDA Zaida CNC University of Coimbra [email protected]

DE LA ROSA Miguel A. University of Seville & CSIC [email protected]

DE MIGUEL VISCASILLAS

Rocío Instituto de Nanociencia de Aragón [email protected]

DE PEREDA José M IBMCC (CSIC-USAL) [email protected]

DIAS Pedro Faculdade de Ciências da Universi-dade de Lisboa

[email protected]

299



DíAZ José Fernando

CSIC [email protected]

DíAZ QUINTANA Antonio Univesidad de Sevilla y C.S.I.C. [email protected]

DIEZ-GARCíA Fernando Instituo de Química-Física Rocaso-lano (IQFR)

[email protected]

DILãO Rui IST, NonLinear Dynamics Group [email protected]

DOBSON Chis M Cambridge university [email protected]

DOMINGO GONZALVEZ Patrícia CQFB - Centro de Química Fina e Biotecnologia

[email protected]

EATON NO VIENE Peter Requimte/University of Porto [email protected]

ECHENIQUE Pablo CSIC [email protected]

EGGERT Helge JPK Instruments [email protected]

ENCISO Marta UNIV. COMPLUTENSE DE MADRID [email protected]

ESPINOSA ANGARICA Vladimir University of Zaragoza [email protected]

ESQUEMBRE TOMé Rocío Universidad Miguel Hernández [email protected]

ESTEBAN MARTíN Santiago Institute for Research in Biomedicine (IRB Barcelona)

[email protected]

ESTRADA Jorge Universidad de Zaragoza [email protected]

FACCIN Mauro BiFi [email protected]

FALCES Jorge Universidad del país Vasco [email protected]

FALO Fernando University of Zaragoza [email protected]

FARIA Tiago Q. Center for Neuroscience and Cell Biology

[email protected]

FELIPE Antonio Universitat de Barcelona [email protected]

FERNANDES Andreia S. IBMC [email protected]

FERNANDEZ-RECIO Juan Barcelona Supercomputing Center [email protected]

FERREIRA NEILA Patricia Universidad de Zaragoza [email protected]

FERRER-MONTIEL Antonio UMH [email protected]

FIASCONARO Alessandro Universidad de Zaragoza [email protected]

FILIPE Luis Carlos Instituto de Tecnologia Química e Biológica

[email protected]

FORNIES BASARTE Aitor Universidad de Zaragoza [email protected]

FRUTOS-BELTRÁN Estrella CSIC-US

FUERST Oliver Universitat Autònoma de Barcelona [email protected]

GALERA Albert Instituto Cajal CSIC [email protected]

GALLEGO Cristina Instituto Química-Física Rocasolano (CSIC)

[email protected]

GAMEIRO Paulo Universitty of Coimbra [email protected]

GARCIA DE LA TORRE José Universidad de Murcia [email protected]

300


GARCIA MANYES Sergi U Colombia, USA [email protected]

GARCíA MAYORAL María Flor Instituto de Química Física Rocasolano, CSIC

[email protected]

GARCIA SEISDEDOS Hector Universidad de Granada [email protected]

GARCíA-ALVAREZ Begoña Centro de Investigaciones Biológicas/CSIC

[email protected]

GARCíA-BARRIOS Alberto Zaragoza University [email protected]

GARCíA-HEREDIA José Manuel Instituto de Bioquímica Vegetal y Fotosíntesis

[email protected]

GARCíA-HERNÁNDEZ Enrique Universidad Nacional Autónoma de México, Instituto de Química

[email protected]

GARCIA-OJALVO Jordi Universitat Politecnica de Catalunya [email protected]

GARCIA-PARAJO Maria IBEC Institute for Bioengineering of Catalonia

[email protected]

GARCIA-SAEZ Ana MPI-MF and DKFZ [email protected]

GARIN Nathalie Leica Microsystems [email protected]

GIERUSZ Leszek University of Warwick [email protected]

GIRALDEZ Teresa HUNSC [email protected]

GIRONA Victoria University of Barcelona [email protected]

GOLLNICK Benjamin Centro Nacional de Biotecnología - CSIC

[email protected]

GOMES Claudio Instituto Tecnologia Quimica e Biologica (ITQB)

[email protected]

GóMEZ María Instituto de BiologíaMolecular y celular del cáncer

[email protected]

GOMEZ FERNANDEZ Juan Car-melo

Universidad de Murcia [email protected]

GOMEZ-MORENO Carlos Universidad de Zaragoza [email protected]

GóMEZ-SICILIA Àngel I. Cajal - CSIC [email protected]

GOMILA Gabriel Institu de Bioenginyeria de Cata-lunya

ggomilapcb.ub.es

GONZÁLEZ SÁNCHEZ María Isabel UNIVERSIDAD DE CASTILLA-LA MANCHA

[email protected]

GOÑI Felix Maria Universidad del Pais Vasco [email protected]

GRACIA LOSTAO Ana Isabel Instituto de Nanociencia de Aragón (Universidad de Zaragoza)

[email protected]

GREGORIO Lucia UNIVERSIDAD MIGUEL HERNÁNDEZ [email protected]

GUALLAR Victor Barcelona Supercomputing Center [email protected]

GUERIN Marcelo Unidad de Biofisica [email protected]

GUERRA Pablo Universidad de zaragoza [email protected]

HENRIQUES Bárbara ITQB/UNL [email protected]

HERGUEDAS Beatriz Universidad de Zaragoza [email protected]

301



HERNÁNDEZ MOLEIRO Lara Universidad Complutense de Madrid [email protected]

HERNÁNDEZ ROCA-MORA

Víctor Manuel

Centro de Investigaciones Biológicas (CSIC)

[email protected]

HERRERO-GALAN Elias IMDEA [email protected]

HERVÁS Ruben Instituto Cajal CSIC [email protected]

HORMEÑO Silvia IMDEA Nanoscience [email protected]

HUARTE Nerea Universidad del Pais Vasco [email protected]

HURTADO-GUERRERO Ramón BIFI [email protected]

IBANES Marta Universitat de Barcelona [email protected]

IBARRA Borja IMDEA Nanoscience [email protected]

IBARRA Ricardo INA [email protected]

JEMAL Imane Universidad de sevilla [email protected]

JESUS Catarina Sofia

University of Coimbra [email protected]

JIMENEZ Mercedes CSIC-CIB [email protected]

JIMéNEZ M. Angeles CSIC [email protected]

JIMéNEZ CABRé Elisa CIB (CSIC) [email protected]

JIMéNEZ ROJO Noemi Unidad de Biofisica [email protected]

KIRAKOSYAN Armen State Engineerng University of Armenia

[email protected]

LAGARTERA Laura Instituto Química-Física Rocasolano [email protected]

LANDAJUELA Ane Universidad del Pais Vasco [email protected]

LANDETA Olatz Univerdad del País Vasco [email protected]

LANS VARGAS Isaias Universidad de Zaragoza [email protected]

LARRIVA María UNVERSIDAD COMPLUTENSE DE MADRID

[email protected]

LASSO CABRERA Gorka Cicbiogune [email protected]

LAURENTS Douglas In. Química Física "Rocasolano" [email protected]

LECTEZ Benoit Unidad de Biofisica [email protected]

LILLO M Pilar CSIC [email protected]

LIN Yibin Universitat autonoma de barcelona [email protected]

LOPES Silvia Universidad do Porto [email protected]

LOPEZ Laura University of Zaragoza [email protected]

LOPEZ MONTERO Ivan Universidad Complutense de Madrid [email protected]

LOPEZ PEREZ Miriam UNIVERSIDAD MIGUEL HERNÁNDEZ, ELCHE

[email protected]

LOPEZ RODRIGUEZ Elena UCM [email protected]

LóRENZ FONFRíA Víctor Universitat Autònoma de Barcelona [email protected]

LOSTAO Maria Pilar Universidad de Navarra [email protected]

LOURA Luís Univ. Coimbra [email protected]

302


LOUSA Diana ITQB-UNL [email protected]

LUGO VéLEZ Carlos Antonio

Universitat Politecnica de Catalunya [email protected]

LLORCA Oscar Spanish National Research Council [email protected]

MAESO GALLEGO Rubén BIOPHYSICS UNIT CSIC-UPV [email protected]

MAGALHãES Pedro ITQB-UNL [email protected]

MANUEL Miguel IBB/CBME (Institute for Biotechno-logy and Bioengineering/Centre for Molecular and Structural Biomedici-ne) - University of Algarve

[email protected]

MARIJUAN Pedro I+CS [email protected]

MARIN Arturo UCM [email protected]

MARTEL Fátima Faculty of Medicine of Porto [email protected]

MARTEL Paulo CBME-IBB [email protected]

MARTíN DE AGUILERA Adriana Instituto Madrileño de Estudios Avanzados (IMDEA)

[email protected]

MARTIN GARCIA Fernando CBMSO-CSIC [email protected]

MARTINEZ Juan UNIVERSIDAD ZARAGOZA [email protected]

MARTINEZ Jesus I Universidad de Zaragoza [email protected]

MARTINEZ-JULVEZ Marta M University of Zaragoza [email protected]

MARTíNEZ-TOMé Mª José Universidad Miguel Hernández [email protected]

MARTINS Ana Paula FFCT-UNL [email protected]

MARTINS Patrícia Faculdade Ciências e Tecnologia Universidade de Coimbra

[email protected]

MAS Francesc Barcelona University [email protected]

MATEO Reyes Universidad Miguel Hernández [email protected]

MATEO VIVARACHO José Angel Universidad de Zaragoza [email protected]

MATESANZ Ruth CIB / CSIC [email protected]

MEDINA Milagros Universidad de Zaragoza [email protected]

MENDIETA Jesus CBMSO-CSIC [email protected]

MENENDEZ Margarita CSIC [email protected]

MENENDEZ Rosa CNB [email protected]

MIGNARD Nicolas wyatt technology [email protected]

MONROY Francisco Universidad Complutense [email protected]

MONTEAGUDO Juan Leica Microsystems [email protected]

MONTERO BARRIENTOS M. Teresa Universidad de Barcelona [email protected]

MONTERROSO Begoña CIB-CSIC [email protected]

MONTES Ruth Biophysics Unit CSIC-UPV/EHU [email protected]

MONTES FERNANDEZ Mª Angeles Universidad de sevilla [email protected]

303



MONTOYA Rocio UNIVERSIDAD DE COLIMA [email protected]

MONTOYA Guillermo CNIO [email protected]

MORAN Federico Universidad Complutense Madrid [email protected]

MORENO Maria João University of Coimbra [email protected]

MORENO Yamir University of Zaragoza [email protected]

MORíN José Alberto Imdea [email protected]

MORO Fernando Universidad del País Vasco [email protected]

MUGA Arturo Universidad del País Vasco [email protected]

MUÑOZ Victor Center for Biological Investigations [email protected]

NAVAJAS Daniel Facultat Medicina - Universitat Barcelona

[email protected]

NAVARRO I GARCIA Aaron IBMC [email protected]

NAVARRO LOPEZ Jorge I+CS jnavarro.iacs@aragon,es

NEHER Margret Universidad de sevilla [email protected]

NIEVA José L University of the Basque Country [email protected]

NOLLMANN Marcelo CNRS [email protected]

OLIVEIRA COSTA Sara Dinisa Universidad Politécnica de Cartage-na (UPCT)

[email protected]

OREL Mikhail Universidad Autònoma de Barcelona [email protected]

ORLANDI Javier Universidad de Barcelona [email protected]

OROZ Javier Cajal Institute/CSIC [email protected]

OROZCO Modesto Universidad de Barcelona [email protected]

ORTEGA PORTERO Esther Centro de Investigación del Cáncer (Universidad de Salamanca-Consejo Superior de Investigaciones Cien-tíficas)

[email protected]

OSPINA RAMIREZ Olga Lucia Universidad Complutense de Madrid [email protected]

PADRóS Esteve Universitat Autònoma de Barcelona [email protected]

PAN Yunzu Academia Sinica [email protected]

PARRA Elisa Universidad Complutense de Madrid [email protected]

PASCUAL José Ignacio Freie Universität Berlin [email protected]

PASTOR DEL CAMPO Isabel universidad de barcelona [email protected]

PEÑA Alvaro CNB (CSIC) [email protected]

PERA Benet CIB / CSIC [email protected]

PEREIRA Manuela Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa

[email protected]

PEREIRA Inês Cardoso ITQB/UNL [email protected]

PEREZ BERNA Ana Joa-quina

Centro Nacional de Biotecnología-CSIC

[email protected]

PéREZ CARRASCO Rubén Universitat de Barcelona [email protected]

304


PEREZ DORADO Inmaculada Laboratory of Molecular Biology

Medical Research Council

[email protected]

PEREZ GIL Jesus Universidad Complutense [email protected]

PEREZ NAVARRO Montse Universidad de Zaragoza [email protected]

PéREZ-CALVO María Angeles

CNB-CSIC [email protected],es

PEREZ-LARA Angel University of Murcia [email protected]

PICOREL Rafael EEAD-CSIC [email protected]

PIE Juan Universidad de Zaragoza [email protected]

PINHEIRO Teresa University of Warwick [email protected]

PONS PEREZ Carles Barcelona Supercomputing Center [email protected]

PRAT Josefa University of Barcelona [email protected]

PRIETO Manuel Centro de Quimica Fisica Molecular [email protected]

PUJOL Jonai UNAV [email protected]

QUERALT-MARTIN Maria Universitat Jaume I [email protected]

RAJA Sebastian IQFR, CSIC [email protected]

RAMíREZ CATAPANO Elisa Universidad Complutense de Madrid [email protected]

RAMOS HERNÁNDEZ Isbaal Biophysics Unit CSIC-UPV/EHU [email protected]

REY Antonio Universidad Complutense de Madrid [email protected]

REYES Nicolas Weill Cornell Medical College [email protected]

RIAL Eduardo Centro de Investigaciones Biológicas - CSIC

[email protected]

RINO José L. Instituto de Medicina Molecular [email protected]

RITORT Felix Universitat de Barcelona [email protected]

RIVAS German CIB CSIC [email protected]

RIZO Josep UT Southwestern Med. Ctr. [email protected]

RODRíGUEZ Mar CSIC [email protected]

RODRíGUEZ ARRONDO José Luís Universidad del País Vasco [email protected]

RODRIGUEZ BUITRAGO Jhon Alexander

UPV [email protected]

RODRIGUEZ CANTALA-PIEDRA

Inma Universitat Politècnica de Catalunya [email protected]

ROMãO Maria João Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa

[email protected]

ROMERO-ROMERO Maria Luisa Universidad de Granada [email protected]

ROQUE Alicia UAB [email protected]

ROUZIC Lionel wyatt technology [email protected]

RUIZ LAZA Rocío University of Seville [email protected]

305



SALMERON-SANTIAGO Karina ENCB, IPN [email protected]

SALVADOR Armindo Center for Neuroscience and Cell Biology

Universitty of Coimbra [email protected]

SALZ Mabel GE Healthcare [email protected]

SÁNCHEZ Ana University of Zaragoza [email protected]

SANCHEZ-RUIZ José Manuel Universidad de Granada [email protected]

SANCHO José M Univ Barcelona [email protected]

SANCHO Javier Universidad de Zaragoza [email protected]

SANCHO MATEO Carlos UNIVERSITY OF NAVARRA [email protected]

SANTOS Nuno Instituto de Medicina Molecular [email protected]

SANTOS Andrea IMM [email protected]

SANZ Joaquin University of Zaragoza [email protected]

SEGOVIA ROLDAN Margarita Universidad de sevilla [email protected]

SEGURA Rosa De Lima

UNIDAD DE BIOFISICA [email protected]

SERNA Marina Centro Nacional de Biotecnología (C.N.B.-C.S.I.C.)

[email protected]

SERRANO Soraya Instituto de Química Fisica Roca-solano

[email protected]

SERRANO Ana University of Zaragoza [email protected]

SHAMIYAN Alina SEUA [email protected]

SILVA DE SOUZA Hileia Karla Universidad Miguel Hernández [email protected]

SIMIC Ana Univeristy of Navarra [email protected]

SIMóN Rosana Universitat Autònoma de Barcelona [email protected]

SOTOMAYOR PéREZ Ana Cristina Institut Pasteur [email protected]

SOUSA Isabel Facultade de ciencias da universida-de do porto

[email protected]

SOVERAL Graça FFUL and Requimte, UNL [email protected]

SUAREZ GERMA Carmen Universidad de Barcelona [email protected]

SUKIASYAN Astghik State Engineering University of Armenia

[email protected]

TABARES Lucia University of Seville [email protected]

TAVARES Bárbara Faculdade de Ciências da Universi-dade de Lisboa

[email protected]

TEIXEIRA Miguel Instituto de Tecnologia Química e Biológica

[email protected]

TORREBLANCA GON-ZALES

Renzo Enmanuel

UNIVERSIDAD DE ZARAGOZA [email protected]

306


TORRENTS Gloria GE Heathcare [email protected]

TORRES BENITO Laura University of Seville [email protected]

TREPAT Xavier University of Barcelona / IBEC [email protected]

TRIGILI Chiara CIB / CSIC [email protected]

UROSEV Dunja UAB [email protected]

USON Alejandro Universidad de Zaragoza [email protected]

VALBUENA Alejandro Cajal Institute/CSIC [email protected]

VALERO RUIZ Edelmira UNIVERSIDAD DE CASTILLA-LA MANCHA

[email protected]

VALPUESTA José CSIC [email protected]

VECINO Ana Julia Unidad de Biofísica [email protected]

VEGA-SANCHEZ Sonia Instituto BIFI [email protected]

VELAZQUEZ-CAMPOY Adrian Instituto BIFI [email protected]

VELEZ Marisela CSIC [email protected]

VERA Andres Manuel

Instituto Cajal CSIC [email protected]

VIEU Christophe INSA/CNRS [email protected]

VILLANUEVA LLOP Raquel Universidad de Zaragoza [email protected]

YRUELA Inmaculada CSIC [email protected]

ZHU Ling CIC biomaGUNE [email protected]

ZORRILLA LóPEZ Silvia CSIC [email protected]

iv spanish portuguese biophysical congress · iv spanish portuguese biophysical congress program...

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