lipoproteins and membranes - embl hamburg · solution scattering from biological macromolecules...
TRANSCRIPT
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LipoproteinsLipoproteins andand MembranesMembranes
P.P.LaggnerLaggner
LectureLecture atat
EMBOEMBO Practical CoursePractical Course on on
SolutionSolution Scattering from Biological MacromoleculesScattering from Biological Macromolecules
Hamburg September 2001Hamburg September 2001
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Institute of Biophysics and X-ray Structure ResearchAustrian Academy of Sciences, Graz, Austria
ÖAW Research Centre Graz
Outstation atElettra / Trieste
Kepler 1596-1600Boltzmann 1870s
Schwarzenegger 1940-
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Lipoprotein groupLipoprotein group::RuthRuth PrasslPrasslRobert SchwarzenbacherRobert Schwarzenbacher
HeinzHeinz Amenitsch Amenitsch Michal HammelMichal Hammel
MonikaMonika ZechnerZechnerSarahSarah TutzTutz
Veronika SattlerVeronika Sattler
JohnJohn ChapmanChapman (INSERM ,Paris)(INSERM ,Paris)
MembraneMembrane groupgroup::KarlKarl LohnerLohnerHeinzHeinz AmenitschAmenitsch
Manfred KriechbaumManfred KriechbaumMichaelMichael RappoltRappolt
Georg PabstGeorg PabstRichard Richard KoschuchKoschuch
Cilaine TeixeraCilaine TeixeraMarlene StroblMarlene Strobl
Monica Monica VidalVidal
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MapMap ofof the Fieldthe Field
TechnaliaTechnalia
BiomedistanBiomedistan
MembraneMembrane
IslandIslandLipoproteinLipoprotein
ArchipellagoArchipellago
SeaSea ofof BiophysicsBiophysics
Cell BiologyCell Biology OceanOcean
CholesterolCholesterolCityCity
Pump Pump StationStation
BilayeronBilayeron
ReceptionReception
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Lipoproteins are Lipoproteins are supramolecular supramolecular particlesparticles inin the the bloodstreambloodstream
Membranes are Membranes are the boundariesthe boundariesofof cellscells andandorganelles organelles
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LIPOPROTEINSLIPOPROTEINS
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VINTAGEVINTAGE
REVIEWREVIEW
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LIPOPROTEIN METABOLISMLIPOPROTEIN METABOLISMNutrition
HDL2
LDL
Chylo-microns
Chylom.Remnants
VLDL
IDL
Extrahepatic
tissues
Macro-phage
Bile acids, Cholesterol
Atherosclerotic
plaques
Foamcell
HDL3
Intestine
EXOGENOUS ENDOGENOUS ATHEROSCLEROTIC
Liver
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LIPOPROTEIN CLASSESLIPOPROTEIN CLASSES
Chylo-micronChylo-micron VLDLVLDL LDLLDL HDL2 HDL3HDL2 HDL3
Density[g/ml] 1.063 1.21
Size[nm] <1000 <80 18-25 8-13
1.00 1.019
LipoproteinFraction
LipoproteinFraction
PlasmaPlasma
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Chem.Comp.
[%]
Apolipo-proteins
PRNPLFCTGCE
CBAIAIIE
CHEMICAL COMPOSITIONCHEMICAL COMPOSITIONChyloChylo--micronmicron VLDLVLDL LDLLDL HDLHDL
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Size [nm]
Den
sity
[g/m
l]0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.3010 20 30 40 50 10 0007060
HDL
LDLVLDL CHYLO-
MICRONS
LIPOPROTEIN PARTICLESLIPOPROTEIN PARTICLES
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Electron microscopyElectron microscopy:: quasispherical particlesquasispherical particles
SAXS:SAXS: what is the internal structurewhat is the internal structure ??
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HighHigh Density LipoproteinsDensity Lipoproteins(HDL)(HDL)
They are the smallest onesThey are the smallest ones ,, their main their main components are proteinscomponents are proteins andandphospholipids phospholipids
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••TheThe SAXSSAXS curvescurvesof all HDLof all HDL are are closely similarclosely similar..
••SideSide maxima maxima indicate indicate quasiquasi--spherical shapespherical shape
••The scaling factorThe scaling factor xxis their reciprocal is their reciprocal sizesize
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p ( r ) p ( r ) –– functionfunction
RadialRadial electron electron density distributiondensity distribution
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HDLHDL CoreCore –– Shell Model Shell Model
•• Cholesteryl estersCholesteryl esters ((aboutabout 3.7 nm3.7 nm longlong)) are radially orientedare radially oriented inin the corethe core..
•• PhospholipidsPhospholipids ((aboutabout 2.2 nm) and2.2 nm) and cholesterol interdigitate from the cholesterol interdigitate from the surfacesurface.. TheThe polarpolar surface layer is aboutsurface layer is about 1.5 nm1.5 nm thickthick..
••Interdigitation is the size limiting principleInterdigitation is the size limiting principle of HDLof HDL
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LOW DENSITY LIPOPROTEIN (LDL)LOW DENSITY LIPOPROTEIN (LDL)
••They contain aboutThey contain about 50% of50% of cholesterolcholesterol ((freefreeandand as fatty acid esteras fatty acid ester))
••Only one apoprotein copyOnly one apoprotein copy perper particleparticle:: apoapo--B, aB, a glycoprotein whichglycoprotein which has ahas a molecular molecular weightweight ofof aboutabout 550550 kD kD
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CholesterylesterCholesterylester
ProteinProteinapoapo B100B100
PhospholipidPhospholipid
CholesterolCholesterol
LOW DENSITY LIPOPROTEIN (LDL)LOW DENSITY LIPOPROTEIN (LDL)
TriglycerideTriglyceride
Old Old ModelModel
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LDLLDL showsshows aa pronouncedpronounced, reversible, reversible transition transition aroundaround 2020--35 °C 35 °C
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fromfrom 3.73.7-- nmnm repeatrepeat totoisotropicisotropic
Concentrical twoConcentrical two--shell shell arrangementarrangement ofof cholesteryl cholesteryl estersesters
OldOld modelmodel
LDLLDL Core StructureCore Structure ::
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LDLLDL--Core below the transitionCore below the transition: :
••inin which orientation are the cholesteryl which orientation are the cholesteryl esters arrangedesters arranged ??
••Fatty acidsFatty acids ofof thethe secondsecond layerlayer inin oror out ?out ?
AA task fortask for SANS !SANS !
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SANSSANS:: selective deuterationselective deuteration ofofcholesteryl esterscholesteryl esters in LDLin LDL
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Analysis ofAnalysis ofGuinier radiusGuinier radius
HH22O/DO/D22O O --
Contrast Contrast variationvariation
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RRxx fromfrom aa comparison between deuteratedcomparison between deuterated andandundeuterated sampleundeuterated sample
oror,, betterbetter,, from the whole contrast from the whole contrast variation series variation series
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Typical Guinier Typical Guinier plotsplots of SANS of SANS onon selectively selectively deuterateddeuterated LDL LDL
ImportantImportant::Concentration Concentration dependencedependence !!
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Models A,B, and CModels A,B, and Cwould agree with would agree with the resultsthe results..
CholesterolCholesterol out /out /
Fatty acidsFatty acids in in
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Which one is correctWhich one is correct ??
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None completelyNone completely !!
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The core transition temperature varies between The core transition temperature varies between individualsindividuals andand dependsdepends onon thethe CE/TG ratioCE/TG ratio
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It showsIt shows aadiscontinuity around discontinuity around
CE / TGCE / TG ratiosratios of 7of 7
This is the core This is the core shellshell ratio in a ratio in a inin two two concentric concentric spheres with spheres with rrii//rroo of 2:1 of 2:1
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The innermost coreThe innermost core(1/8 of(1/8 of the core the core
volumevolume)) retainsretains aafluidfluid,, oily state oily state even below the even below the
transitiontransition
The cholesteryl The cholesteryl esters phaseesters phase
separateseparate into theinto thesecondsecond layerlayer
T<TmCE/TG>7
Tmhigh
T<TmCE/TG<7
Tmlow
T>TmT G
C E
F C
P L apo B100
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THE END :THE END :
NeitherNeither SAXSSAXS nornorSANSSANS can can differentiate between differentiate between the two modelsthe two models
ButBut:: modelmodel BB fits the fits the data fromdata from DSC and DSC and ESRESR
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Can the core transition follow Can the core transition follow the physiological temperature the physiological temperature changeschanges inin the blood streamthe blood stream??
AA task for synchrotron task for synchrotron radiationradiation SAXSSAXS
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Temperature [°C]
10 20 30 40 50
Excess Heat Capacity
Tm
Liquid crystalline Isotropic oily16 - 32°C
DSC and SAXS
CORE LIPID TRANSITIONCORE LIPID TRANSITION
0.5
10°C
40°C
20x103
15
10
5
0Inte
nsity
(a.
u.)
0.40.30.20.10.0
s (1/nm)
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TIMETIME--RESOLVED XRESOLVED X--RAY DIFFRACTIONRAY DIFFRACTIONKinetics of core transition
Melting: Laser T-jump10ms time resolution
time [ms]s [1/nm)
inte
nsity
[a.u
.]
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Freezing of core lipids
Temperature drop: 40°C 10°C250 ms time resolution
0
5
10
0.3
0.15
0.45
time
[s]
s [1/nm]
inte
nsity
[a.u
.]
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Melting isMelting is fast: onfast: on the millisecondthe millisecond timetime scalescale
Freezing isFreezing is onon the timescalethe timescale of sec, i.e. fastof sec, i.e. fastenoughenough toto follow thefollow the TT--changeschanges inin slowly slowly exchanging vesselsexchanging vessels
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From structure analysisFrom structure analysis totosynthesissynthesis::
LDLLDL asas drugdrug carriercarrier
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drug intercalation in core lipids
drug intercalation in surface monolayer Problems of incorporation:
drug intercalation results in a disordering of the core region and changes in apoB-100 conformation
Drug incorporation in LDL
C
O
CH2O
HO
OPO
CH2OO
-CHNH
CHOH
COC17H35
15H31
NHCH3
O N
5'-ceramide thymidine (CET)
O
CH3
3',5'-dioleyl thymidine(DOT)
CO
CH3(CH2)7CH:CH(CH2)7 O
OCO
CH3(CH2)7CH:CH(CH2)7
CH2O
NO
NH
O
H3
5'-monooleyl deoxythymidine(MOT)
CO
CH3(CH2)7CH:CH(CH2)7 OCH2O
NO
CNH
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Characterisation of the LDLCharacterisation of the LDL -- DOT DOT drug complexes drug complexes with with SAXSSAXS
Experimental SAXS curves from LDL below the phase
transition
0 5 10 15 20 25
LD L con tro l 20 .4±0 .1 nm
LD L na tive 20.2±0.4 nm
LD L-D OT 21 .5± 0.4 nm
p(r
)
r [nm]
• The peak maximum at large distances for native LDL was rmax 20.2±0.4 nm, which corresponds to the electron density autocorrelation of the phospholipid headgroups and protein moiety.
• Broadening of maximum peak for LDL control without significant difference in rmax value indicate formation of LDL aggregates during incubation.
• Increase in rmax value (∆r=1.3±0.6 nm) and broadening of peak maximum for LDL-DOT indicate slightly increase in the maximum particle diameter and formation of LDL aggregates.
Real space electron-pair distance distribution
functions
0.0 0.5 1.0 1.5 2.0 2.5 3.0
LDL native LDL control LDL-DOT (5 DOT molecules per LDL)
log
I(h)
h [nm-1]
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Characterisation of the LDLCharacterisation of the LDL--MOT MOT drug complexes drug complexes with with SAXSSAXS
Experimental SAXS curves from LDL below the phase
transition
• No significant differences have been observed in rmax value of peak maximum for native, reconstituted LDL as also for LDL-MOT complex with 50 molecules of drug per LDL particle.
• Incorporation of MOT have no significant effect on particle diameter and core lipid arrangement
Real space electron-pair distance distribution
functions
0.0 0.5 1.0 1.5 2.0 2.5 3.0
LDL native LDL control LDL-MOT (50 MOT molecules per LDL)
log
I(h)
h [nm-1]0 5 10 15 20 25
19.6 nm20.2 nm20.0 nm
LDL native LDL reconstituted LDL-MOT (50 MOT molecules per LDL)
p(r)
r [nm]
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The StepThe Step to highto high resolutionresolution::
LDLLDL CrystallographyCrystallography
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Images taken at EMBL Hamburg
LDLLDL crystalscrystals
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ImagesImages takentaken atatElettraElettra: 180 ° rotation : 180 ° rotation inin stepssteps of 10°/of 10°/frameframe
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CrystallographyCrystallographye.m., X-ray diffraction3D protein structure
ESR Spin LabellingESR Spin Labellinglipid mobilityorder parameter and polaritylipid-protein-interaction
SAXSSAXS
size and shapeinternal organization
DSCDSCprotein stabilitythermotropiccore-melting
core-transitionTm=24-31°C
Triglyceride
Cholesterylester
Free Cholesterol
Phospholipid
Protein (apo B100)
PHYSICAL METHODSPHYSICAL METHODS
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MEMBRANES
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Structure Dynamics
Thermodynamics
FunctionFunctionFunction
Non-Equilibrium
WHAT A MESS !
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Starting from the components:
Lipid self-assembly
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LIPID POLYMORPHISM
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A typical T/c -phase diagram
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A little bit of theory...
(Biologists may doze now for a while, and dream of Monica)
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Uncorrelated bilayers / Vesicles
Continuous scattering
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The solution:
Electron density profile
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Multilayer stack
Liposome
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Bilayer Stacks / Liposomes
Bragg - reflections
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Multilayer stack
Liposome
Swelling series
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Small- and wide-angle scattering
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This can also be done with natural membranes, e.g. erythrocyte ghosts
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Unilamellar vesicles and multilamellar liposomes can coexist
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Catching up speed, slowly.
Temperature scanning
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main
transition
Lβ
Lα
maintransition
pre-transition
Lβ’
Pβ’
Temperature
Pressure
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Equilibrium StructuresSlow T-scan 500 sec / frame (sealed tube 2kVA)
small angle wide angle
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Equilibrium ?
NO
Synchrotron T-scan
medium speed
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Phase epitaxy
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Physiological relevance(Biologists wake up)
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LHC complex shifts L/H equilibrium in reconstituted membranes towards bilayer
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Excess lipid is stored as hex-phase
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Getting faster – chasing intermediates
T/p jumps
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Head of Project: Peter Laggner1) [email protected] Contact: Heinz Amenitsch1)* [email protected]
igrid Bernstorff 2) [email protected]: Pavo Dubcek2) [email protected]
Michael Rappolt1)* [email protected]: Cristian Morello1) [email protected]
1) Institute for Biophysics and X-ray Structure Research (IBR) 2) Sincrotrone Trieste Austrian Academy of Sciences Strada Statale 14, km 163.5 Steyrergasse 17, 8010 Graz, Austria. 34012 Basovizza (TS), Italy.Tel 0043-316-812 004 Tel 0039-040-375 81 Fax 0039-040-938 0902 Fax 0043-316-812 367 *) Working site: IBR c/o Sincrotrone Trieste
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DYNAMICSDYNAMICSIRIR--LASER TLASER T--JUMPJUMP
IR-Laser:λ = 1.54 µmEmax = 4 Jτ = 2 ms∆Tmax = 20°C
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Intermediate also in lam/hex transition
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ShortShort--lived Intermediatelived Intermediate
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Lαααα *Lα Lα
Non-Equilibrium StructureOrdered Intermediate Phase
0 5 10 15
6.2
6.3
6.4
6.5
d (n
m)
t (s)
30 40 50 60 70
6.40
6.45
6.50
T (°C)
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lateral expansion
com
pres
sion
T-jump:1-2 ms
∆T = 5 - 15 °C
Relaxation:~ 30 - 40 s
Lαααα Lαααα*
Re-diffusion of water
T-jump probes the elastic properties of liposomes
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Milos Steinhart(IMC Prague)
Heinz Amenitsch (IBR)Sigrid Bernstorff (Sincrotrone Trieste)
High-pressure SAXS:Lipids and Cholesterol
Monica Vidal (IBR)Cilaine Teixera (IBR)Manfred Kriechbaum (IBR)
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0 5 10 15 20 25
0,004
0,006
0,008
0,010
0,012
0,014
0,016
0,018
0,020
0,022
|∆
d|/d
eq
xC (mol %)
Effects of Cholesterol on Bilayer ElasticityT-jump induced change in lamellar repeat as a function of cholesterol content in POPC
HardeningSoftening
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Reference: M.Steinhart, M.Kriechbaum, K.Pressl, H.Amenitsch, P.Laggner and S.Bernstorff, Rev.Sci.Instrum. 70, 1540-1545 (1999).
The HP-X-ray Cell
Cell body: Stainless steel (5 x 3.5 x 2 cm)
Max. pressure: 2-3 kbar (5-90°C)
Windows: Be or diamond C (2r = 3 mm, d=1.5 and 0.75 mm, respectively)
Aperture: entrance: 1.5 mm, exit: 2 mm (2r); 60° angle cone (covers SAXS and WAXS).
Sample: optical path: 1.5-3 mm.
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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6 POPC 20% + 1% mol cholesterol, T=20°C
d [n
m]
P [Kbar]
time
[s]
scattering vector
335 bar
1782 bar
300 bar
0
220
P-scans of POPC (20%) with different cholesterol concentrations (0/1/5/10/20 mol%) at different
temperatures (5/10/15/20°C)
Pm
Pm
110 frames, 2s/frame
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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6 POPC 20% T=20°C
d [n
m]
P [Kbar]
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6POPC 20% + 5% mol cholesterol, T=20°C
d [n
m]
P [Kbar]
0.0 0. 2 0.4 0.6 0.8 1.0 1.2 1.4 1. 6 1.8 2. 06.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6 P OPC 20% + 1% mol cho lestero l, T=20°C
d [n
m]
P [Kbar]
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6POPC 20% + 10% cholesterol, T=20°C
d [n
m]
Pressure [Kbar]
Hysteresis for POPC 20% and different molar concentrations of cholesterol at 20°C d-spacing as a function of pressure during a p-scan (red: compression and blue: decompression)
0 % 1 %
5 % 10 %
Pressure-Hysteresis
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Transition pressure Pm of 20% POPC at different molar cholesterol concentrations (0-20%) asa function of temperature obtained from pressure-scans followed by SAXS.
Clausius-Clapeyron slopes
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dP/dT
0 5 10 15 2045
50
55
60
65
70
dP/d
T
% cholesterol0 2 4 6 8 10
-40
-30
-20
-10
0
10
20
30
40
20°C 15°C 10°C 5°C
hyst
eres
is/ra
te
% cholesterol
Rate-dependence of hysteresis
Both, Clausius-Clayperon slopes (dP/dT) and hysteresis/rate show a maximum at low concentrations of cholesterol (1-5 mol%). This agrees with the notion that low amounts of
cholesterol induce a softening/plastification and higher concentration cause hardening.
Pressure scanning of PC / Cholesterol mixtures
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0 5 10 15 20 25
0.050
0.055
0.060
0.065
0.070
0.075
0.080
dP/d
T
xc (mol %)
0 2 4 6 8 10 12 14 16 18 200.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
delta
Pm
xc (mol %)
Transition pressure Pressure hysteresis T-jump induced change of d-spacing
Pabst G., Rappolt M., Amenitsch H., Bernstoff S., and Laggner P. (2000). Langmuir 16, 8994-9001
Comparison of p-Scan and T-jump Results
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20% POPC, 0.33M LiCl, 15°C 25% POPC, 0.33M LiCl, 15°C
Lαααα*Lαααα*L ααααL αααα
L ααααL αααα
Lαααα*Lαααα*
L αααα /Lαααα* - Equilibrium Phase Separation by Li+-Ions
Pressure stabilizes L αααα
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Surface Diffraction
φ
θΟ
θΙ
Scattered beam
sample
Incident beam
In-Plane structure
Depth structure
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Surface Diffraction Cell
M y la r
H 2 O
L ip id s
S i
X - r a y s
25 µµµµm~50 µµµµm
500 µµµµm
Type I:
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Surface Diffraction
Lββββ „Gel-Phase“
Lαααα „Liquid crystalline-Phase“
6 nm
Fig.: Surface diffraction pattern of SOPE heated from 20 - 40 - 20 ºC with a rate of 1ºC/min showing the diffraction peaks from the2nd to the 6th order at fixed incidence angle ω (1.2º). The upper resolution limit was just given by the dimension of the vacuum tube and the detector length. The phase transition Lβ - Lα- Lβ (@ 35 ºC) is clearly visible.
Problem: Absorption and angular correction!!Poiseuille shear cell for neutrons: Hamilton et.al., Physica B 221, (1996) 309-319
Interface correction factor and Cross-section correction factor
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Film deposition & microscopyFilm deposition & microscopy
Spray coating „air brush“
Air brush: IWATA HP-A as microdispensersystem, max volume 1ml
• constant N2 pressure 0,4bar
Microscopy picture (160x magnification)of 10mg/ml POPC / isopropanol solution,deposited on Si-wafer
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Film deposition & microscopyFilm deposition & microscopy
Spin coating
Microscopy picture (160x magnification)of 10mg/ml POPC / isopropanol solution,deposited on Si-wafer
Transpipette: TRANSFERPETTOR, max volume 50µlDrilling machine: BOSCH CSB550
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POPC/ 10mg/ml POPC/ 10mg/ml (from (from isopropanolisopropanol) full hydration ) full hydration
spray coating (air brush)spray coating (air brush)
Image plate Omega – scan : x-axis = s-scaley-axis = omegaz-axis = intensity
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DPPC: dry, at partial and fully hydration:DPPC: dry, at partial and fully hydration:
Sample No 8: 10 mg/ml DPPC/isopropanol on Si-wafer (spin-coated)
dry partial hydrated fully hydrated
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LiCl Concentration d (Å) Multilayer Peak d (Å) Isotropic Ring
0M 63.1
0.10M 64.6
0.33M 64.8 57.8
0.55M 65.09 56.0
0M, after removal of LiCl 63.9
Lithium / Lipid - Interaction In-Situ
0.1M 0.33M
0.5M 0M
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POPC at full hydrationPOPC at full hydration
Sample No 23 – spin-coated
Image plate Omega – scan : x-axis = s-scaley-axis = omega
z-axis = intensity
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High Pressure Surface Diffraction
Entrance Nipple
IncidentX-ray beam
Diamond Windows
Pressure Media
Silicon Waver
Sketch of the Surface Diffraction High Pressure Cell
10 mm
Specularreflectedbeam
•X-ray energy: 16 keV
•Diamond windows: 0.75 mm thick
•Beam size: 0.5 x 0.2 mm2
•max incidence angle: 4°
•exit aperture: 60° total
•sample surface: 3 x 3 mm
•p : 0-3 kbar
•temperature range: 0-80 °C
Fig.: set-up
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High Pressure Surface Diffraction
Fig.: Diffraction pattern of DMPC in the Lα Phase at 30 °C during the ω-scan. FWHM of ω-scan: < 0.03°.
Fig.: Diffraction pattern at 30 °C:p-scan (1 -
1736 bar) Lα/Pβ’/Lβ’
DMPC (Lββββ ’/Pββββ ’/Lα α α α @ 15/25 °C)
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High Pressure Surface Diffraction
Fig.: Diffraction pattern of POPE at 25 °C/1 bar
Tim
e (s
)
s (1/nm)
POPE (Lββββ/Lαααα transition at 22 °C)
POPE: p-scan (1000 - 1 bar, Lβ/Lα transitionat 280 bar)
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WHERE DO WE GO FROM HERE ?
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NanocompositesNanocomposites at Interfacesat Interfaces
Solid-state Soft (bio)materials
Bio-inspired design of functional devices
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Specific Recognition
Semiconductor
NANOBIONICS : MEMBRANE ON CHIP
Sensitive Amplification
Electronics
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SolidSupport
Mechano- Electronic Handling
Protein
Specificity Triggering
Lipid
Sensitivity
COST D-22: WG Membranes at Surfaces
Institute of Biophysics and X-ray Structure Research Austrian Academy of Sciences http://www.oeaw.ac.at/ibr/
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IBR / Elettra Heinz AmenitschSigrid Bernstorff Pavo DubcekMichael Rappolt Georg Pabst
IBR / Graz: Manfred Kriechbaum, Ruth Prassl,Ingrid Winter, Karl Lohner,Richard Koschuch, Monica Vidal,
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