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A selection of interesting papers that were published inthe two months before our press date in major journalsmost likely to report significant results in chemical biology.

Current Opinion in Chemical Biology 2000, 5:91–99

Contents (chosen by)

91 Proteomics and Genomics (Matthews and Tewari)91 Biocatalysis and biotransformation (Pohl)92 Bio-inorganic chemistry (Cammack)93 Combinatorial chemistry (Hall)94 Next generation therapeutics (Projan)96 Analytical techniques (Cass)96 Mechanisms (Stewart)97 Model systems (Roberts and Sanders)98 Biopolymers (Flitsch, Lowden and Newman)

• of special interest•• of outstanding interest

Proteomics and genomicsSelected by Lisa Matthews and Muneesh TewariDana-Farber Cancer Institute, Boston, USA

A functional genomics strategy that uses metabolome datato reveal the phenotype of silent mutations. Raamsdonk LM,Teusink B, Broadhurst D, Zhang N, Hayes A, Walsh MC,Berden JA, Brindle KM, Kell DB, Rowland JJ et al.: Nat Biotechnol2001, 19:45-50.•• Significance: This paper shows that an approach based onmeasurement of relative changes in metabolite concentrationscan reveal phenotypes for gene deletions that otherwise conferno overt phenotype. The results suggest that gene function maybe deciphered by comparing metabolite profiles from strainsdeleted in such ‘silent’ genes with those from strains havingdeletions of genes with known cellular roles.Findings: The authors made use of the principle that metabo-lite concentrations are more sensitive to changes in enzymaticactivity than are overall metabolic fluxes (as reflected by growthrate, for example). By measuring levels of metabolites in yeaststrains carrying deletions of metabolic enzymes that are ‘silent’with respect to differences in growth rate, they identified dis-tinct metabolite profiles corresponding to different classes ofdeletion mutant strains. Proof of principle experiments usingNMR spectroscopy showed that the approach can be general-ized to the case in which the metabolites to be measured areunknown. Sophisticated analysis and clustering of the NMRspectra of cell extracts from mutant strains allowed them to beplaced into distinct groups without any a priori knowledge ofthe specific metabolites being analyzed.

Functional annotation of a full-length mouse cDNA collection.The RIKEN Genome Exploration Research Group Phase II Teamand the FANTOM Consortium: Nature 2001, 409:685-690.• Significance: This paper describes the isolation, sequencingand chromosomal mapping of a large collection of mousecDNAs representing an estimated 13,000 unique genes. Thisis an important resource for future functional genomic studies.

Findings: The authors used approximately 160 cDNA librariesprepared by various methods, including normalization andmRNA cap-trapping technologies, to isolate cDNA clonesenriched for full-length and novel transcripts. Sequencing of theclones identified many novel genes as well as new proteinmotifs and families. Gene annotation was carried out as part ofan internationally organized functional annotation meeting andthe sequences and annotation are available via the World WideWeb (http://genome.gsc.riken.go.jp).

The protein–protein interaction map of Helicobacter pylori.Rain J-C, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S,Lenzen G, Petel F, Wojcik J, Schachter V et al.: Nature 2001,409:211-215.• Significance: This paper describes a large-scale protein–pro-tein interaction map of the gastric pathogen H. pylori using 261bacterial proteins as baits in yeast two-hybrid screens. Detailsof the strategy used vary somewhat from those of other map-ping projects of this sort and may offer advantages, particularlyfor analyses of prokaryotic genomes.Findings: The authors used a high-throughput version of theyeast two-hybrid assay to search for interacting proteins for261 different predicted open reading frames from H. pylori.Over 1200 interactions were identified. The authors argue thattheir strategy of utilizing a prey cDNA library made from randomgenomic fragments recovers more interactions and offers theopportunity for a robust domain analysis in many cases.

Arabidopsis transcription factors: genome-wide compara-tive analysis among eukaryotes. Riechmann JL, Heard J,Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O,Ratcliffe OJ, Samaha RR et al.: Science 2000, 290:2105-2110.• Significance: This paper describes the first genome-wide com-parison of transcriptional regulators in eukaryotic organisms.Findings: This paper reveals that the similarity among C. ele-gans, Arabidopsis and Drosophila families of transcriptionfactors is limited to the DNA-binding domain and that consid-erable domain shuffling has contributed to a high degree offunctional diversity of these proteins.

Biocatalysis and biotransformationSelected by Nicola PohlIowa State University, Ames, USA

New approach to multiply deuterated isoprenoids usingtriply engineered Escherichia coli and its potential as a toolfor mechanistic enzymology. Kakinuma K, Dekishima Y,Matsushima Y, Eguchi T, Misawa N, Takagi M, Kuzuyama T,Seto H: J Am Chem Soc 2001, 123:1238-1239.•• Significance: Metabolically engineered organisms can pro-vide means to biosynthesize deuterated and other labelednatural products that would be difficult to obtain by chemicalsynthesis. In addition to enhancing studies on the possiblefunctions of these natural products, these labeled compoundscan also stimulate insight into the mechanistic enzymologyinvolved in these pathways. Findings: E. coli was engineered to switch all isoprenoid syn-thesis from a non-mevalonate pathway to a mevalonatepathway. All isopentenyl diphosphate (IPP) in the engineered

Chemical biologyPaper alert

organism must be derived from exogenously fedmevalonate-pathway intermediates. The genes for the biosyn-thesis of zeaxanthin were also introduced into the bacterialstrain. Upon addition of mevalonolactone-d9, a partially deuter-ated zeaxanthin was isolated. Protons were discovered atzeaxanthin carbons derived from biosynthetic intermediatesthat would be protonated during the isomerization of dimethy-lallyl diphosphate (DMAPP) to IPP.

ββ-Mannosynthase: synthesis of ββ-mannosides with amutant ββ-mannosidase. Nashiru O, Zechel DL, Stoll D,Mohammadzadeh T, Warren RAJ, Withers SG:Angew Chem Int Ed Engl 2001, 40:417-420.• Significance: The β-D-mannopyranoside linkage is found in avariety of complex carbohydrate structures, but is one of themost challenging to chemically synthesize. The enzymes thatmake these linkages are now available, but require an expensiveand complex nucleotide diphosphate donor. A mutant β-man-nosidase is shown to carry out the desired reaction using asimple α-D-mannosyl fluoride as a donor.Findings: The active site nucleophilic glutamine of the retainingβ-mannosidase cloned from Cellulomonas fimi was convertedto a serine to produce a ‘glycosynthase’. This mutant enzymelost its ability to cleave mannoside linkages and was able tosynthesize β-D-mannopyranosides using mannosyl fluoridesand a variety of glycosyl acceptors. Overall yields ranged from71% to 99%, but mixtures of multisaccharides were producedfrom the addition of more than one mannose residue.Surprisingly, a fluoride ion could act as a nucleophile in thechemical rescue of mannoside-cleavage activity of the mutantmannosidase in addition to the traditional anionic nucleophilessuch as acetate, azide, and formate.

Cloning, sequencing and analysis of the enterocin biosynthesisgene cluster from the marine isolate ‘Streptomyces maritimus’:evidence for the derailment of an aromatic polyketide synthase.Piel J, Hertweck C, Shipley PR, Hunt DM, Newman MS, Moore BS:Chem Biol 2000, 7:943-955.• Significance: Aromatic polyketide synthase gene clustersprovide the code for the biosynthesis of several clinically impor-tant compounds such as doxorubicin. Previously sequencedgene clusters of this class have included DNA sequences forcyclases or aromatases that are responsible for the formation ofthese aromatic compounds. A newly sequenced gene clusterencoding the biosynthetic pathways for the enterocin andwailupemycin polyketides lacks such genes, which may in partaccount for the diversity of compounds produced. Findings: A 21.3 kilobase gene cluster encoding the biosynthe-sis of the enterocin and wailupemycin families of polyketides froma marine Streptomyces was cloned and sequenced. Twenty openreading frames were discovered that included genes for thebiosynthesis of the relatively unusual benzoyl-coenzyme A starterunit as well as a gene encoding for a putative oxygenase respon-sible for a Favorskii-like rearrangement.

Bio-inorganic chemistrySelected by Richard Cammack King’s College London, London, UK

Mechanistic diversity in a metalloenzyme superfamily.Armstrong RN: Biochemistry 2000, 39:13625-13632.•• Significance: Structural genomics provides new insightsinto the functional relationships between apparently unre-lated metalloproteins. It is proposed that a group of enzymes,

characterized by a similar arrangement of paired βαβββmotifs, but containing different metal ions, represent a super-family of metalloproteins. This structure permits the metal ionto have two or three open coordination sites and consider-able catalytic versatility. Findings: The proposed vicinal oxygen chelate (VOC) super-family comprises enzymes that bind their substrates orreaction intermediates via a pair of oxygen atoms. They includeglyoxalase I (containing Zn2+ or Ni2+); the fosfomycin resis-tance protein (containing Mn2+); and the extradiol dioxygenase(containing Fe2+). Another enzyme, methylmalonyl-CoAepimerase (containing Co2+) binds its substrate by a non-vici-nal oxygen atoms.

The catalytic center in nitrous oxide reductase, Cu-z, is a cop-per-sulfide cluster. Rasmussen T, Berks BC, Sanders-Loehr J, Dooley DM, Zumft WG, Thomson AJ: Biochemistry 2000,39:12753-12756.

ANDRevisiting the catalytic CuZ cluster of nitrous oxide (N2O)reductase: evidence of a bridging inorganic sulfur atom.Brown K, Djinovic-Carugo K, Haltia T, Cabrito I, Saraste M,Moura JJ, Moura I, Tegoni M, Cambillau C: J Biol Chem 2000,275:41133-41136.• Significance: Nitrous oxide reductase catalyses the final stepof bacterial denitrification, in which it reduces the greenhousegas N2O to N2. The enzyme contains a unique four-coppercluster, CuZ. New spectroscopic evidence, and re-examinationof the crystallographic data, indicate that the four copper atomsin the CuZ cluster are bridged by a sulfide atom in an unprece-dented type of metalloprotein centre.Findings: The crystal structure of N2OR shows two copperclusters. CuA is µ-oxo dimeric cluster similar to that incytochrome c oxidase; and the CuZ cluster comprises four cop-per ions, bound by seven histidines, in a distorted tetrahedralarrangement around a single central ligand atom. This atomwas originally modeled as an OH-. However, magnetic circulardichroism, confirmed by resonance Raman spectroscopy,showed the presence of sulfur coordination to the copper. Thiswas demonstrated to be a sulfide by analysis of the protein, andby refinement of the structure of Paracoccus denitrificansN2OR at 160 pm resolution.

Identification of the haemoglobin scavenger receptor.Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman HJ,Law SKA, Moestrup SK: Nature 2001, 409:198-201•• Significance: Hemoglobin is released from erythrocytesby hemolysis, particularly in autoimmune diseases, infec-tions such as malaria, and inherited conditions such assickle-cell anemia. It would be a source of toxic oxygen rad-icals if it were not complexed by haptoglobin and eliminatedby tissue macrophages. The protein in macrophage mem-branes, that is responsible for receptor–mediatedendocytosis, has been identified. Findings: The receptor protein CD163 was isolated by chro-matography with a hemoglobin–haptoglobin affinity matrix.CD163 binds the complex of the two proteins tightly andselectively in a Ca2+-dependent process. Chinese hamsterovary cells transfected with CD163 cDNA acquired the abilityto take up the hemoglobin–haptoglobin complex. CD163, hap-toglobin, and heme oxygenase in macrophages comprise ahemoglobin clearance system that is up-regulated by agentsthat induce inflammation.

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The 70-kDa major DNA-compacting protein of the chloro-plast nucleoid is sulfite reductase. Sato N, Nakayama M,Hase T: FEBS Lett 2001, 487:347-350.• Significance: The nucleoids of chloroplasts are complexes ofchloroplast DNA and proteins, which are capable of replicationand transcription. An unexpected finding is that the siroheme-iron–sulfur protein sulfite reductase is a component of thecomplex that is needed for folding of DNA.Findings: A 70 kDa nucleoid protein was identified as sulfitereductase by sequencing and by immunoblotting with a spe-cific antibody. The enzyme isolated from nucleoids of peachloroplasts contained siroheme, as seen by its spectrum, andwas fully active as a ferredoxin:sulfite reductase. The enzymeappears to be a structural component but its catalytic functionis uncertain, because ferredoxin is not present in the nucleoid.

Direct FeS cluster involvement in generation of a radical inlysine 2,3-aminomutase. Cosper NJ, Booker SJ, Ruzicka F,Frey PA, Scott RA: Biochemistry 2001, 39:15668-15673.•• Significance: Lysine 2,3-aminomutase is one of a group ofenzymes that perform many free-radical reactions for degrada-tion of compounds under anaerobic conditions. Theyincorporate a glycine radical, which is generated by a reactionwith S-adenosyl methionine. X-ray spectroscopy of an interme-diate in the reaction of the enzyme indicated that a direct bondis formed between the methionine sulfur and an iron–sulfurcluster. The formation of radicals in this way represents a noveltype of reactivity of iron–sulfur clusters.Findings: Selenomethionine was used as an analogue ofmethionine that could be probed by selenium K-edge X-rayabsorption fine structure (EXAFS) spectroscopy. The complexof lysine 2,3-aminomutase with a substrate analogue and Se-adenosyl selenomethionine was examined as a model of thetransition state. A scattering peak at 0.27 nm from the seleniumwas assigned to an iron atom of a [4Fe–4S] cluster. A pro-posed mechanism for the enzyme, which is probably applicableto other glycine radical enzymes, is that the S-adenosylmethio-nine is cleaved to form a complex of methionine with theiron–sulfur cluster, and a 5′-deoxyadenosyl radical.

Combinatorial chemistrySelected by Dennis HallUniversity of Alberta, Edmonton, Alberta, Canada

A synthetic library of cell-permeable molecules. Koide K,Finkelstein JM, Ball Z, Verdine GL: J Am Chem Soc 2001,123:398-408.• Significance: Previous approaches to promote protein dimer-ization have involved known ligands with well-characterizedmacromolecules. There is significant interest in the generationof synthetic libraries of cell-permeable small molecules that canact as heterodimerizers for unknown or weakly characterizedproteins and effect novel cellular responses in intact cells.Findings: To test this new concept, a library of diverse het-erodimers was designed with a constant half based onAP1867, a known ligand for the FK506-binding protein (FKBP).The other invariant half, chosen on the basis of various aspectssuch as ease of synthesis and amenability to combinatorialdiversification, is the tetrahydrooxazepine framework (THOX).The latter was constructed using a combination of liquid-phaseand solid-phase methods, which led to the synthesis of a320-membered library of potential heterodimerizers of FKBPand diverse intracellular targets. In order to employ the librarytowards intracellular targets, it is of utmost importance that the

compounds be capable of crossing the cellular membrane.Toward this end, a representative sample of 25 library memberswas tested and eventually found to be fully permeable tofibrosarcoma cells at a concentration of 100 nM. This work isconsidered a first but crucial step toward using THOX-basedlibraries of heterodimerizers in cellular assays.

Catalyst screening using an array of thermistors.Connolly AR, Sutherland JD: Angew Chem Int Ed 2000,39:4268-4271.•• Significance: It is very important to improve the capability forevaluating libraries of potential biological or chemical catalystsin parallel, with high accuracy, and within a short timeframe. Thedevelopment of multiplexed arrays of highly sensitive thermis-tors appears to represent a significant improvement overprevious technologies such as infrared thermography.Findings: A thermistor array apparatus of 0.1 milliKelvin preci-sion was made. Its use toward the relative quantitation ofdifferent catalytic species was first tested with the β-lactamasehydrolysis of penicillin at different concentrations of enzyme.This enzymatic reaction is moderately exothermic. Nonetheless,because of the sensitivity of this apparatus, the values of maxi-mum temperatures observed were found to correlatesatisfactorily with enzyme concentration. Conversely, inhibitionof β-lactamase by sodium clevulate under successive dilutionsprovided data similar to a conventional titration curve, suggest-ing the potential use of this method for the discovery of newenzyme inhibitors. Finally, enantioselective alkene epoxidationby a cobalt (II)-salen catalyst was also investigated as an exam-ple of non-biological catalysis.

Nonpeptidic ααvββ3 integrin antagonist libraries: on-beadscreening and mass spectrometric identification withouttagging. Gibson C, Sulyok GAG, Hahn D, Goodman SL,Hölzemann G, Kessler H: Angew Chem Int Ed 2001,40:165-169.• Significance: Split-pool synthesis is a powerful combinato-rial strategy for the rapid generation and screening of largebead-supported libraries. When applied to non-peptidic mol-ecules, however, one usual limitation of this approach is therequirement for bead-encoding strategies. This work shows,for the particular class of peptidomimetics described, thatfragment analysis from mass spectrometry of single beadscan be successfully employed to identify library membersfrom unencoded libraries.Findings: Two 330-membered diacylhydrazine libraries ofArg–Gly–Asp (RGD) peptidomimetics, based on the aza-glycine replacement, were synthesized by a split-poolsynthesis scheme using four linear substructural modulesA–D. A respective number of 6, 3,10 and 2 non-isobaric build-ing blocks were selected for each module of these libraries,which were assembled on TentaGel macrobeads. Analysis ofmodel sequences by electrospray ionization mass spectrome-try (ESI-MS) had shown that unambiguous compoundidentification is possible based on the decomposition patternaround the diacylhydrazine moiety. The libraries were thenincubated against biotinylated αvβ3 integrin receptor, onemember of a family of therapeutically relevant targets known tobind to RGD peptides. A secondary enzymatic assay for stain-ing hit beads revealed a small number of promisingsequences. The tightest binder showed an IC50 of 0.15 micro-molar, and displayed some selectivity over two other integrinreceptors. According to the authors, this work represents the

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first successful application of an unencoded one-bead-one-compound library of non-peptidic molecules.

Computer aided analysis of split and mix combinatoriallibraries. Topiol S, Davies J, Vijayakuma S, Wareing JR:J Comb Chem 2001, 3:20-27.• Significance: Deconvolution strategies are required in orderto identify hits from unencoded split-pool, ‘one bead-one-mole-cule’ libraries of small organic compounds. This requiresintensive efforts of selective resynthesis for portions of thewhole library. Computational analyses can help optimize thechoice of an appropriate splitting and mixing scheme withrespect to the particular characteristics of the library. Findings: A computational approach examined possible pat-terns of splitting and mixing for several interrelated parameters:total library size, number of combinatorial steps, number ofreaction vessels and number of compounds in the final pools.The method potentially allows other factors and constraints tobe added at will (e.g. tagging operations, cleavage, etc.). Theresulting analysis revealed that the splitting/mixing strategy hassignificant impact on the overall efficiency of deconvolution andresynthesis efforts.

Combinatorial approach to the development of fluorescentsensors for nanomolar aqueous copper. Singh A, Yao Q, Tong L,Still WC, Sames D: Tetrahedron Lett 2000, 41:9601-9605.• Significance: The development of molecular sensors formetal ions is a problem of high importance in environmentalmonitoring and toward the study of intracellular processes. Asshown in this work, semi-rationale combinatorial strategies canbe of tremendous help in identifying selective ligands for transi-tion metal ions in water. Findings: A 1470-membered split-pool library of ionophoresbased on three triazaalkane scaffolds (two cyclic, one acyclic)was elaborated by diversifying aminoalkyl sidechains with sevenamino acids and a variety of heterocyclic capping groups.Screening of the library against copper ions using a colorimet-ric assay allowed the identification of several promising ligands.One of them, a bis(pyrazine)-containing ligand derived from the1,4,7-triazacyclononane scaffold, was further studied. It was re-synthesized and shown to be very selective for copper (II) ionsin the presence of other transition metal ions. This and othersensors featuring simple chemical modifications were attachedto polymeric microspheres. Their use in an optical fiber systemallowed the detection of a broad range of copper ion concen-trations down to nanomolar range.

Identification and isolation of a receptor for N-methyl alkylam-monium salts: molecular amplification in a pseudo-peptidedynamic combinatorial library. Cousins GRL, Furlan RLE,Ng Y-F, Redman JE, Sanders, JKM: Angew Chem Int Ed 2001,40:423-428.• Significance: By combining the concepts of molecular evolu-tion and combinatorial chemistry, dynamic combinatoriallibraries (DCLs) can facilitate the identification of receptors forsmall organic molecules. This study suggests the feasibility ofcovalent DCL approaches for the discovery of new functionalmolecules such as catalysts and therapeutic drugs. Findings: A very simple model receptor system was designedbased on the assembly of a bifunctional benzaldehyde-cappedproline-based hydrazide monomer. Whereas mainly the cyclicdimeric or trimeric macrocycles are formed under acid-pro-moted hydrazone exchange, the cyclic dimer is predominant in

the free mixture in chlorofom. In the presence of either acetyl-choline (ACh) or N-methylquinuclidinium (NMQ) cations, aftera two-day equlibration, product distribution is strongly affectedthrough apparent templating with the ligand. The trimerspecies was highly amplified according to HPLC–electrosprayionization mass spectrometry (ESI-MS). Overall, the resultsobtained constitute a 50-fold amplification relative to the cyclicdimer. The ACh and NMQ trimer complexes were further stud-ied by MS and multidimensional nuclear magnetic resonanceto obtain approximate binding constants and insights on theirstructural characteristics.

Multi-step organic synthesis using solid-supportedreagents and scavengers: a new paradigm in chemicallibrary generation. Ley SV, Baxendale IR, Bream RN,Jackson PS, Leach AG, Longbottom DA, Nesi M, Scott JS,Storer RI, Taylor SJ: J Chem Soc Perkin Trans 12000:3815-4195.• Significance: The use of solution-phase synthesis with thehelp of solid supported reagents and scavengers has becomea very attractive option for the generation of small-moleculelibraries with potential pharmaceutical and agrochemical appli-cations. A comprehensive review of this field is long overdue,and will surely help guide chemists in the synthetic design ofnew libraries.Findings: The authors of this 280-page review compiled over2000 articles. The contents are well organized, with a multitudeof tables describing the different types of supported reagentsand scavengers.

Next generation therapeuticsSelected by Steven ProjanWyeth-Ayerst Research, Pearl River, New York, USA

Pharmacokinetics, safety and antiviral effects of hypercin, aderivative of St. John’s wort plant, in patients with chronichepatitis C infection. Jacobson JM, Feinman L, Liebes L,Ostrow N, Koslowski V, Tobia A, Cabana BE, Lee DL,Spritzler J, Prince AM: Antimicrob Agents Chemother 2001,45:517-524.• Significance: Over 100 million people worldwide are chroni-cally infected with the hepatitis C virus (HCV). HCV, apestivirus, is a significant cause of morbidity and mortality andit is the largest single cause of liver transplants. Currently, thebest therapy is a combination of ribavirin and interferon-alphabut response rates are in the range of 40–50%. Therefore,there is need for more efficacious and less toxic therapies forthe treatment of HCV. It had been previously reported that asmall molecule natural product from the St John’s wort plant,named ‘hypericin’, displayed antiviral activity in vitro against sev-eral viruses including bovine diarrhea virus (BVDV), which issimilar to HCV. However, the hypericin activity against BVDVwas only manifest in the presence of light. Nevertheless, aphase I, escalating dose clinical trial using hypericin inHCV-infected patients was attempted.Findings: Twelve patients received an eight-week course oftherapy consisting of orally administered hypericin once daily at0.05 mg/kg body weight; seven patients were given 0.10mg/kg once daily. Five of 12 patients at the lower dose and sixof seven at the higher dose experienced phototoxicity, but noother adverse effects were reported. Relatively long half-lives of36.1 and 33.8 hours, respectively, were noted for the twogroups and reasonable levels of exposure (mean area under thetime-concentration of 1.5 and 3.1 µg/ml hr) were observed.

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However, plasma HCV titers showed no difference during thecourse of the study (untreated or placebo-control patients werenot included in the data set nor were patients undergoing morestandard HCV therapy). These were undertaken in partbecause of the lack of good in vitro culture systems for HCVand the reliance upon BVDV as a surrogate. The justification forthe study is that St John’s wort extract is used safely by manyself-medicating individuals for a variety of purposes and, there-fore, there was little risk to the HCV patients in the study.However the fact that activity of hypericin against BVDV wasonly observed in the presence of light led to predictable results.

Cross-resistance between triclosan and antibiotics inPseudomonas aeruginosa is mediated by multidrug effluxpumps: exposure of a susceptible mutant strain to triclosanselected nfxB mutants over-expressing MexCD-OprJ.Chuanchuen R, Beinlich K, Hoang TT, Becher A, Karkhoff-Schweizer RR, Schweitzer HP: Antimicrob Agents Chemother2001, 45:428-432.•• Significace: Triclosan may be the most-used antimicrobialagent in the 21st century so far. It is found in pajamas, cuttingboards, toothpastes, hand soaps and deodorants to name afew examples. Previously (McMurry LM, Oethinger M, Levy SB:Nature 1998, 394:531-532 1998), it was reported that FabI(an enzyme, enoyl-acyl carrier protein reductase, involved infatty acid biosynthesis) was the molecular target of triclosan inEscherichia coli. Ironically, the Levy group was expecting to findresistance based on selection of strains over-expressing so-called ‘multidrug efflux pumps’ (mdrs). Yet there is littleevidence of triclosan selecting for cross-resistance to otherantibacterial agents in E. coli (a twofold decrease in triclosansusceptibility was observed in AcrAB over-expressing strain indata published by the Levy lab).Findings: Selection for triclosan-resistant strains in P. aerugi-nosa was apparently trivial, a mutation frequency of 10–6 wasobserved in strain PAO1, and of the small number of sponta-neous mutants sampled all exhibited an mdr phenotypeshowing concomitant decreases in susceptibility to tetracy-cline, ciprofloxacin, erythromycin and trimethoprim. Westernblot analyses revealed over-expression of the MexCD-OprJ mdrproteins and nucleotide sequencing demonstrated that nfxB,which encodes a regulatory protein, was mutated, suggestingan increase in mexCD-oprJ transcription. These findings, takentogether with those of McMurry et al., demonstrate that selec-tion for a given resistance mechanism is not uniform among allbacteria and points to the ease with which multidrug-resistantstrains can be selected in P. aeruginosa. Specifically, theseresults indicate that, as we increase the environmental expo-sure of P. aeruginosa to triclosan, we can expect to find moreclinical isolates resistant to therapeutic agents that would nom-inally treat infections caused by that bacterium.

Antibiotic activity and characterization of BB-3497, a novelpeptide deformylase inhibitor. Clements JM, Beckett RP,Brown A, Catlin G, Lobell M, Palan S, Thomas W, Whittaker M,Wood S, Salama S et al.: Antimicrob Agents Chemother 2001,45:563-570.•• Significance: Antimicrobial drug resistance has stimu-lated interest in finding and exploiting novel bacterial targetsfor the discovery and development of new antibacterialagents. Towards that end, genes encoding essential func-tions and which are widely conserved among pathogenicbacteria have been the subject of drug discovery programs.

Peptide deformylase (PDF) is a metalloenzyme that deformy-lates the N-formylmethionine residue of nascentpolypeptides and is considered an essential function(although a gene knockout of the def gene encoding peptideformylase can be obtained in a genetic background defectivein the formyl transferase gene, fmt) as reported by Margoliset al. (Antimicrob Agents Chemother 2000, 44:1825-1831).Given that there is no equivalent human enzymatic activity,PDF has been the subject of several screening projects toidentify specific inhibitors as antibacterial leads.Findings: A library of compounds focusing on those withmetal-chelating properties was used to screen for inhibition ofPDF activity. Of the several compounds identified in thescreen, a natural product antimicrobial, actinonin, a hydrox-amic acid, was identified. Consistent with this finding, Chen etal. (Biochemistry 2000, 39:1256-1262) recently demon-strated that actinonin is, indeed, a potent Staphylococcusaureus PDF inhibitor. The authors also reported that a syn-thetic compound, BB-3497, was also a potent inhibitor of PDF(7 nm IC50 vs Escherichia coli PDF in vitro compared with a10 nM IC50 for actinonin). BB-3497 showed structural simi-larity to actinonin as well as to fMet–Ala–Ser, a recognizedsubstrate of PDF. In vitro assays showed modest antibacterialactivity for BB-3497 4–16 µg/ml against strains of S. aureus;8 µg/ml against a wild-type E. coli strain. Actinonin was lessactive but did show some antibacterial activity. Resistance toBB-3497 was reported (although not the mutation frequency),the resistant strains in S. aureus and E. coli that were analyzedall have defects in the respective fmt genes with these mutantstrains somewhat compromised in terms of slower growth invitro. Whether an antimicrobial agent that only impairs ratherthan inhibits growth (or kills bacteria) will be a successfulantibiotic is an open question.

The structural basis for the action of antibiotics tetracy-cline, pactamycin, and hygromycin B on the 30S ribosomalsubunit. Brodersen DE, Clemon WM, Carter AP,Morgan-Warren RJ, Wimberly BT, Ramakrishnan V: Cell 2000,103:1143-1154.•• Significance: The bacterial ribosome is one of nature’sfavorite antimicrobial targets. Several antibiotics including themacrolides, chloramphenicol, the tetracyclines, and the amino-glycosides act on ribosomes and inhibit protein synthesis. Inseveral instances, especially for the tetracyclines, the moleculartarget for the inhibition of protein synthesis was only conjecturalalthough the available data indicate that tetracyclines inhibitpeptide chain elongation by blocking the entry of aminoacyltRNAs into the acceptor (A) site on the 30S ribosomal subunit.One of reasons for this difficulty in determining the moleculartarget of antibiotics that target the ribosome is that few target-based mutations have been identified for ribosomally activecompounds. Rather, dedicated resistance determinants areobtained by horizontal gene transfer. In the case of the tetracy-clines, these function to either reduce intracellular tetracyclineconcentration by efflux or to protect the ribosome by bindingdistally to the tetracycline-binding site causing a more rapid dis-sociation of tetracycline from its target.Findings: The authors soaked crystals of the Thermus ther-mophilus 30S ribosomal subunit with three differentantibiotics: tetracycline, pactamycin and hygromycin B. Thelocation of the antibiotics within the 30S subunit was deter-mined by X-ray diffraction. In general, the locations of theantibiotics were consistent with “much but not all biochemical

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data”. In fact, two binding sites were identified for tetracy-cline, the more frequently occupied site is located at the Asite and the hydrogen bonding of the molecule with the ribo-some at this site defines precisely the portion of thetetracycline molecule, which cannot modified without losingantibacterial activity. “The less occupied site is at the inter-face of three RNA domains in the body of the subunit.” Inboth instances, no proteins are involved in tetracycline bind-ing; this is in contrast with two earlier studies. Pactamycin,contrary to previous data, appears to interact with the exit (E)site, resulting in a 12.5 angstrom displacement and affectingmRNA entry/positioning. Hygromycin B is an aminoglycosideantibiotic that has been shown to block translocation and, tosome extent, cause misreading of mRNA. The data demon-strated a single hygromycin B binding site near the A, E andpeptidyl (P) sites and where other aminoglycosides havebeen reported to bind. The importance of this work and otherstudies now elucidating the structure of the ribosome cannotbe minimized. The science is compelling in and of itself andthe potential utility in finding or improving upon compoundsthat interact with the ribosome is great.

Analytical techniquesSelected by Tony Cass Imperial College of Science, Technology and Medicine, London, UK

A high signal-to-noise Ca probe composed of a singlegreen fluorescent protein. Nakai J, Ohkura M, Imoto K: NatBiotechnol 2001, 19:137-141.• Significance: The real-time, non-invasive measurement ofintracellular concentrations of key species is an importanttool in molecular cell biology. One of the most intensivelystudied intracellular analytes is calcium and considerableeffort has gone into producing improved reagents for cal-cium imaging. In this paper, a derivative of greenfluorescent protein (GFP) that can be used for calciumimaging is described.Findings: A fusion protein comprising a cyclically permutedGFP (cpGFP) with calmodulin (CaM) at the carboxyl termi-nus and the M13 fragment of the myosin light chain at theamino terminus was constructed. When calcium binds toCaM, the latter interacts with M13 causing a conformationalchange in cpGFP that in turn alters the fluorescence ofcpGFP. The fusion protein showed an affinity for calcium of200 nM and its use in measuring calcium in HEK-239 cellsand myotubes was demonstrated.

Interference-based detection of nucleic acid targets onoptically coated silicon. Jenison R, Yang S, Haeberli A, PoliskyB: Nat Biotechnol 2001, 19:62-65.•• Significance: Nucleic acid hybridisation methods arewidely used in biological research and clinical diagnostics.Many variants of the basic hybridisation method have beendescribed but nearly all require labelling of the probe or tar-get nucleic acid. This adds considerably to the complexity ofthe method and this paper describes a simple visual determi-nation of nucleic acid duplexes.Findings:At the heart of the method is a crystalline silicon sur-face to which oligonucleotide probes are bound. The colour oflight reflected from the surface is very sensitive to the thicknessof the top layer, shfting from gold to red as it increases from 0Å to 100 Å. The detection limit for DNA using this device isaround 0.1 attomole and its application to the detection of anantibiotic resistance gene (mecA).

Model-based analysis of oligonucleotide arrays: expressionindex computation and outlier detection. Li C, Wong WH:Proc Natl Acad Sci USA 2001, 98:31-36.•• Significance: DNA microarrays are becoming standardtools for global analysis of gene expression and other applica-tions involving nucleic acid hybridisation. Although highdensities of oligonucleotides offer a powerful method for mas-sively parallel analysis there are issues associated withprobe-dependent bias, cross hybridisation and array contami-nation. A model-based statistical approach to dealing withthese issues is presented.Findings: The authors present a simple model that analysesperfect match/mismatch probe hybridisation intensities and cantake account of outliers as well as artefacts such as scratches.The application of these techniques is demonstrated with largeexpression arrays for human and murine genes.

MechanismsSelected by Jon D StewartUniversity of Florida, Gainesville, Florida, USA

Novel catalytic mechanism of nucleophilic substitution byasparagine residue involving cyanoalanine intermediaterevealed by mass spectrometric monitoring of an enzymereaction. Ichiyama S, Kurihara T, Li Y-F, Kogure Y, Tsunasawa S,Esaki N: J Biol Chem 2000, 275:40804-40809.• Significance: This is a remarkable example of how thechemical course of an enzyme mechanism can change to sur-mount an active site mutation that would otherwise appear topreclude catalysis.Findings: The normal mechanism of Pseudomonas sp.YL L-2-haloacid dehalogenase involves an SN2 displacement ofchloride by the sidechain of Asp10 to form an ester intermedi-ate that is subsequently hydrolyzed to give the 2-hydroxyacid.Surprisingly, the mutant in which Asp10 was converted to Asn(D10N) retained a low level of catalytic activity that was not dueto deamidation of Asn10. Mass spectrometry was used to mon-itor the dehalogenation of L-2-chloropropionate by the D10Nmutant. The data were consistent with SN2 displacement ofchloride by the Asn10 sidechain to form an imidate from whichlactic acid was eliminated to produce a (b)-cyanoalanineresidue at position 10. The active-site nitrile was partiallyhydrolyzed to regenerate the amide sidechain of Asn10, pre-sumably by the same catalytic machinery normally responsiblefor hydrolyzing the ester intermediate.

Strain is more important than electrostatic interaction incontrolling the pKa of the catalytic group in aspartateaminotransferase. Mizuguchi H, Hayashi H, Okada K,Miyahara I, Hirotsu K, Kagamiyama H: Biochemistry 2001,40:353-360.• Significance: The fact that individual protein residues playmultiple roles in structure and function often complicates theinterpretation of site-directed mutagenesis experiments. Thisstudy describes a way to address this issue using an amino-transferase as an example.Findings: Aspartate aminotransferase is a well-studiedtransaminase that utilizes a pyridoxal phosphate cofactor that isbound to the sidechain of Lys258 via a Schiff’s base in the rest-ing form of the enzyme. Substrate binding increases the pKa ofthe Schiff’s base nitrogen by 2.0 pH units and this is critical infacilitating the transaldimination process, which yields an iminewith the (a)-amine of the donor amino acid, the first intermedi-ate in transamination. Two active-site guanidinium groups,

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contributed by Arg292 and Arg386, were clearly important inthis process; however, the precise way in which these tworesidues increased the pKa of the Schiff’s base was not clear.Using a series of site-directed mutants, the manner by whichthese two arginine residues increase the pKa by a combinationof electrostatic effects, domain motion and Schiff’s base con-formational strain were elucidated.

Transition state structure of purine nucleoside phosphory-lase and principles of atomic motion in enzymatic catalysis.Fedorov A, Shi W, Kicska G, Fedorov E, Tyler PC, Furneaux RH,Hanson JC, Gainsford GJ, Larese JZ, Schramm VL, Almo SC:Biochemistry 2001, 40:853-860.•• Significance: A transition-state mimic designed from theexperimental transition state structure was shown by X-ray crys-tallography to mimic many of its steric and electronic propertiesincluding ground state destabilization and enzyme movement.Findings: Purine nucleoside phosphorylase catalyzes the dis-placement of purine nucleoside bases by phosphate to yieldthe free base and the (a)-anomer of ribose-1-phosphate. Kineticisotope effect studies supported an oxocarbenium-like transi-tion state. Immucillin-H was designed to mimic this species bya protonated pyrroldine ring with a C–C bond to a 9-deazahy-poxanthine ring, which itself is protonated on N7. Comparingthe X-ray crystal structures of free and enzyme-bound immu-cillin-H revealed that the inhibitor conformation was distortedupon binding. Specifically, the pucker of the pyrrolidine ringwas altered significantly and the angle between the hypoxan-thine and pyrrolidine rings was flattened. These distortions ofthe inhibitor mirror the steps necessary for the normal substrateto reach the transition state.

Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basisfor its remarkable catalytic promiscuity. Polekhina G,Board PG, Blackburn AC, Parker MW: Biochemistry 2001,40:1567-1576.• Significance: The crystal structure of this enzyme shows bothintriguing similarities and differences with other glutathione S-trans-ferases and provides the first step toward explaining the diversity ofchemical reactions observed for this remarkable enzyme.Findings: Human maleylacetoacetate isomerase catalyzes twophysiologically significant glutathione-dependent reactions: acis-trans olefin isomerization that is the penultimate step ofphenylalanine and tyrosine degradation and the hydrolysis of1,1-dichloroacetate to glyoxalate. X-ray crystallography showedthat the structure of the human enzyme was similar to other glu-tathione S-transferases and the position of bound glutathionewas consistent was its mechanistic roles proposed for bothcis–trans isomerization and dichloroacetate hydrolysis.Although the sequence of the human isomerase/glutathionetransferase is very similar to that of the bacterial enzyme tetra-chlorohydroquinone dehalogenase, this similarity apparentlydoes not extend to the catalytic role of a conserved cysteineresidue (Cys16) that is essential for the bacterial enzyme butnot for the human protein.

Model systemsSelected by Sarah L Roberts and Jeremy KM Sanders University of Cambridge, Cambridge, UK

Development of small peptides recognizing a monosac-charide by combinatorial chemistry. Sugimoto N, Miyoshi D,Zou J: Chem Commun 2000, 23:2295-2296.

•• Significance: Protein-oligosaccharide interactions play akey role in the control of various normal and pathologicalprocesses. Therefore, it is crucial to understand the molecularbasis of these interactions. This paper shows how a combina-tion of design and combinatorial synthesis has led to a modelpeptide system that shows specific recognition of a monosac-charide. The model system incorporates two key bindingfeatures, hydrogen-bonding and Van der Waals interactions.Findings: Alanine-based oligomer peptides with one or two try-tophan residues were synthesised in order to test therelationship between peptide conformation and monosaccha-ride–trytophan indole sidechain van der Waals interactions.Fluorescence measurements in the presence and absence of D-galactose indicated the positions in which the trytophanresidues gave rise to significant monosaccharide recognition.Combinatorial synthesis of a biased peptide library was thenused in an attempt to position both of the binding modes in theappropriate orientation in a small peptide (i.e. van der Waalsinteractions arising from the previously discovered trytophanpositions and hydrogen-bonding from acidic residues in thepeptide chain). From a library of 62,000 peptides, 15 wereshown to have significant selectivity for the target saccharideD-erythrose with surprisingly high binding constants in theregion of 104 M–1. High selectivity is achieved through a com-bination of hydrogen bonding to the saccharide hydroxy groupsand van der Waals packing of the hydrophobic sugar faceagainst aromatic acid sidechains forming what are called ‘sand-wiching interactions’. These results demonstrated the criticalroles of both the aromatic and acidic residues for the multi-pointsaccharide recognition observed.

Identification and isolation of a receptor for N-methyl alkylammonium salts: molecular amplification in a pseudo-peptide dynamic combinatorial library. Cousins GRL,Furlan RLE, Ng Y-F, Redman JE, Sanders JKM: Angew Chem IntEd Engl 2001, 40:423-428.•• Significance: Dynamic combinatorial chemistry offers a newstrategy for the identification of new host/guest compoundsthat are potential enzyme mimics, sensors or transporters. Thisapproach combines the merits of combinatorial chemistry withmolecular evolution, using a combinatorial library of moleculesmade up of various building blocks, which can interconvertthrough reversible bonds between the different building blocks.Thus, the library is under thermodynamic control and may beinfluenced by template effects, molecules binding strongly tothe template becoming amplified, whereas those with poorbinding decrease in concentration. If the template used is atransition-state analogue for a given reaction, the library mem-bers amplified can be identified as potential catalysts. In thispaper, the authors report molecular amplification from adynamic combinatorial library (DCL) of pseudo-peptides of amacrocyclic receptor that binds selectively to acetylcholine(ACh) and N-methylquinuclidinium salts (NMQ). The shifts inproduct distribution reported are the largest yet observed forcovalent dynamic combinatorial systems and serve as proof ofthe concept of thermodynamic control of product distribution inDCLs by template effects. This work sets the scene for theidentification of new more efficient and selective receptors andenzyme mimics.Findings: A DCL was generated from a proline-based buildingblock equipped with hydrazide and aldehyde groups and,therefore, able to form oligomeric pseudo-peptide cyclichydrazones reversibly. The hydrazone exchange was acid

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catalysed and the library was shown to reach thermodynamicequilibrium after two days, giving a mixture comprised mainlyof cyclic dimer. Cyclisation in the presence of the N-methy-lalkylammonium salts NMQ or ACh, gave rise to dramaticallydifferent library compositions strongly biased towards thecyclic trimer. This product distribution was obtained irrespec-tive of whether the template was introduced before theaddition of acid, or to the equilibrating mixture containingmainly dimer. This confirms that the distribution observedarose from a thermodynamically controlled process, not bykinetic trapping of the trimer. The authors demonstrate that theobservations arise from specific template effects, using massspectrometry and 1H NMR spectroscopy to demonstrate theformation of cation : cyclic-trimer adducts.

BiopolymersSelected by Sabine FlitschEdinburgh University, Edinburgh, UK

Glycolipid antigen processing for presentation by CD1dmolecules. Prigozy TI, Naidenko O, Qasba P, Elewaut D,Brossay L, Khurana A, Natori T, Koezuka Y, Kulkarni A,Kronenberg M: Science, 2001, 291:664-667.•• Significance: The data presented here demonstrate a car-bohydrate antigen processing system analogous to that usedfor peptides, and the ability of T cells to recognise processedfragments of complex glycolipids. Findings: The requirement for processing glycolipid antigens inT cell recognition was examined with mouse CD1d-mediatedresponses to glycosphingolipids. Although some disaccharyllipids were recognised without processing, other antigens suchas Gal(α1–2)GalCer, required removal of the terminal sugars topermit interaction with the T-cell receptor. Interestingly, lysoso-mal α-galactosidase A, previously thought to be solely involvedin glycosphingolipid metabolism was identified as the enzymeresponsible for the de-glycosylation step.

Development of an efficient, regio- and stereoselective routeto libraries based on the beta-D-glucose scaffold.Hirschmann R, Ducry L, Smith A: J Org Chem 2000,65:8307-8316.• Significance: Carbohydrates are attractive scaffolds for thegeneration of compound libraries but a drawback has beenthe difficulty of selective functionalisation of hydroxyl groupswith very similar reactivity. This paper describes an efficientroute for the sequential functionalisation of the five hydroxylgroups of glucose.Findings: An efficient synthetic route to orthogonally protectedethylthioglucosides was developed that allowed the synthesisof a small glucose-based library. It is suggested that this strat-egy is amenable to solid-phase synthesis and will facilitate theconstruction of diverse monosaccharide libraries.

Direct isolation and sequencing of specific protein-bindingglycosaminoglycans. Keiser N, Venkataraman G, Shriver Z,Sasisekharan R: Nat Med 2001, 7:123-128.•• Significance: Heparin/Heparan-sulfate-like glycosamino-glycans (HLGAG) play important roles in the regulation ofprotein stability and function. Identification of the precise gly-cosaminoglycan sequences involved in these events has beenvery difficult. This paper demonstrates for the first time howsurface non-covalent affinity mass spectrometry can be usedto rapidly isolate and directly sequence the biologically rele-vant HLGAG oligosaccharides.

Findings: A recently reported method of using matrix-assistedlaser desorption ionisation mass spectrometry to analysepicomole amounts of HLGAG oligosaccharides was com-bined with immobilisation of a HLGAG binding protein on asurface to perform affinity isolation and sequencing of tissue-derived HLGAG oligosaccharides on a mass spectrometersurface. This method of analysis was first tested to study thebinding of known ligands to antithrombin III and was able todistinguish between specific and non-specific binding inter-action, even within a mixture of saccharides. The studies werethen extended to find and identify oligosaccharide sequencesthat bind to the fibroblast growth factor FGF-2 by combiningthe method with other sequencing techniques such as selec-tive HLGAG hydrolysis.

Selected by Philip AS LowdenUniversity of Exeter, Exeter, UK

Metal-ion coordination by U6 small nuclear RNA con-tributes to catalysis in the spliceosome. Yean S-L,Wuenschell G, Termini J, Lin R-L: Nature 2000, 408:881-884.•• Significance: The spliceosome is the RNA–protein assem-bly that excises introns from messenger RNA prior totranslation. An understanding of its molecular mechanism istherefore an important goal in studying the regulation of geneexpression. This study provides the first direct evidence that thespliceosome RNA plays an active role in catalysis, as has alsorecently been convincingly demonstrated for the ribosome.Findings: The authors prepared U6 small nuclear RNA mole-cules carrying a phosphorothioate linkage at U80 andseparated the RP and SP stereoisomers. Reconstitutionyielded complete, correctly assembled spliceosomes. In thepresence of Mg2+ (which normally supports catalytic activity),splicing was inhibited. In the presence of Mn2+, however, amore thiophilic metal ion, splicing activity was restored for theSP isomer only. Mn2+ binding was shown to be competitivewith Mg2+ binding, indicating that sulfur substitution does notcause significant conformational changes. These resultsstrongly suggest that a specific RNA–metal interaction plays arole in spliceosome catalysis.

Nonenzymatic autoligation in direct three-color detection ofRNA and DNA point mutations. Xu Y, Karalkar NB, Kool ET:Nat Biotechnol 2001, 19:148-152.• Significance: This is an important addition to the rapidlygrowing array of techniques for genetic analysis. It pos-sesses important advantages: high sequence-specificity; norequirement for reagents other than hybridising oligonu-cleotides; and efficient recognition of RNA as well as DNA.It is also an elegant example of how atomically precise syn-thetic modification of biopolymers can find importantpractical application.Findings: This work utilises the facile reaction between a3′-phosphorothioate monoester and a 5′-iodothymidine whenthe reacting DNA strands are hybridised to a third templatestrand. The reaction was found to be highly sensitive to DNAmismatches, allowing discrimination of point mutations ashigh as 104-fold. Importantly, RNA could be detected withequal efficiency, something which has not been achievedusing enzymatic ligation. Substrates labelled with fluo-rophores were used to detect ligation by fluorescenceresonance energy transfer. Single base pair mutations couldbe detected readily by labelling wild type and mutant ligationsubstrates with different fluorophores.

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Selected by Richard NewmanImperial Cancer Research Fund, London, UK

Trans-complex formation by proteolipid channels in the ter-minal phase of membrane fusion. Peters C, Bayer MJ,Buehler S, Andersen JS, Mann M, Mayer A: Nature 2001,409:581-588.•• Significance: The mechanism of membrane fusion, withoutwhich all membrane traffic within a cell would grind to a halt, wasthought to be mediated solely by SNARES (proteins that inhabitall fusion-competent membranes of a cell). The authors describe asituation in which SNARES do not cause fusion directly butinstead enable an ion-permeable membrane protein, the vacuolarH+ATPase, to form a bridge between membranes, allowing fusionto proceed. The important implication of these observations is thatthe mechanism of membrane fusion is explained in terms of theradial expansion of a proteolipid-lined protein channel.Findings: The authors show that two yeast vacuoles fuse byforming a head-to-head dimer of the VO part of the vacuolar

proton pump, which is found in most intracellular mem-branes and vesicles. This pump contains two multisubunitsectors — V1, which hydrolyses ATP, and VO, a complex ofsix so-called proteolipids — together with associated sub-units through which proteins are transported. Thedimerisation of VO is proposed to allow the two proteolipidhexamers to form a closed-off channel from one vacuole intothe other, suggesting a mechanism for fusion. Calcium-bound calmodulin is believed to cause the proteolpids toseparate within the plane of the membrane, so that lipidsinvade the spaces between them and the aqueous channelin the centre opens. The separating proteolipids exposebetween them surfaces that have a hydrophobic portionsandwiched between two hydrophilic stripes. Such surfacesmimic lipid bilayers and allow lipids to invade the proteolipidring without leaving their bilayer configuration. This model isattractive because it explains fusion without invoking tempo-rary lipid intermediates.

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