ProteinFoldingBIBC100TheFoldingProblem¢ HowProteinsFold? Consideraproteinwith100a.a.¢ 10100possibleconformations(avg.of10conformations/a.a.)¢ Ifitconvertsfromoneconformationtoanotherin˜10-13secthentheavg.timetosampleallconformationswouldbe1077yearsor1085secondsCosmicTerm:longerthanthelifeofearth/universeLevinthalʼsParadox¢ However,invivo,proteinsfoldin10-1-103seconds,amismatchof>98ordersofmagnitude¢ Conclusion:Foldingisnotrandom‡deterministic(directed)¢ NativeState(foldedstate) Unique(action) Stable(energy) Accessible(kinetics)U.S.A.SecondGeneticCode:Sequence StructureFoldingDesign(reversefolding)SequenceStructureDesignStructureSequenceFoldingOutputInputComputerAlgorithmImportance?1.Toomanyseq.,fewstruct.(105‡106,genome)(<103)Understandingseq.-struct.relationrequiressolving2.BiotechnologyunleasedpowerDesign-drugs,hormones,sensors,processes(photosynthesis)ToomanystructuresorTheFoldingProblemPhysicalForces¢ Hbond(local)<nearneighbors>¢ Hydrophobic(compactness/moltonglobule)<distantneighbors>FoldingPathwaysFunnelsExploretheenergylandscapeorconformationalSpace(degreesoffreedom)CharacteristicsofFoldedState¢ Tightpackingcompact¢ Sequencedetermined/environmentmodulated (N-P)‡SearchSpace¢ Familiesandsymmetry¢ Eachseq.‡uniquestructure¢ Nativestateisthermodynamicallystable(lowestenergy)USADogmaPhysicalForces¢ Hbond(local,nearneighbors)¢ Hydrophobic(compactness/moltonglobule,distantneighbor)FoldingPathwaysFunnelsExploretheenergylandscapeorconformationalspace(degreesoffreedom)ComputationalModeling¢ Majorareaofresearch¢ Infancy WestillcannotfoldproteinsbycomputerNeeded:1. Understandingprocess2. Definingtheminimum3. Fastercomputers4. ModelstestablebyexperimentationThatʼswhyfoldinganddesignaretwodifferentformulationsofthesameproblemInVivoFolding¢ Chaperonesbindtoincompletelyfoldedpolypeptides Preventaggregation Regulatetranslocation¢ FoldasescatalyzefoldingFoldasesN I UchaperonesNativeIntermediateFoldingRxʼs:-DisulfideBonds-x-propeptidebonds-cis-transisomersGoal:Topreventaggregation(collapsedintermediates)andalternativelyfoldedstatesExistenceofFoldingintermediatesdetectedbyNMR¢ Obtainedbyanalysisofthedisulfidebondingpatternofintermediatestrappedduringreoxidationofa59a.a.protein(bovinepancreatictrypsininhibitor)¢ Role:transientstructuresinnascentchainscouldinitiateearlystepsinfolding(funnels)¢ Biotechnologyproblamatic‡inclusionbodiesProteinStability(thermal)¢ Proteinengineering(mutagenesis)1. S-Sbridgesa. -CH2-S-S-CH2-b.Analysisofallpossibilities(many)c.Energyminimizationtoreducetoafewplausiblecandidatesd.Site-selectivemutationse.Proteinsynthesisf.Assay:exampleT4lysozyme(x-raystructureknown)Reducingdegreesoffreedom(entropy)increasesproteinstabilityProteinStabilityCont2.GlyandPro-Gly‡freedom-Pro‡Constraints(sidechainisfixedbycovalentbondtomainchain-Gly‡Prohaspropensitytoincreasestability(moredeficate)-Gly‡Alausuallyincrease-Gly‡usuallydecreaseProteinStabilityCont3.DipolarstabilityN-end(-a.a.)C-end(+a.a.)-increasestabilitybymutatingresiduesatN-endofhelicesfrompolartonegative(e.g.ASN‡ASP,SER‡ASP)Helix{ProteinStabilityCont4.Hydrophobicityinthecone(cavity)-Barnase(bacterialRNAse-110a.a.)-structurebybothx-rayandNMR-introducingcavitiesintheconebymutationssuchasIle‡ValorPhe‡LeuCavityforaCH2Stabilityby1kcal/mol-Moredelicatedesign-NeedsstructurePredictionofStruct.FromSeq.¢ Empiricalinprogress¢ ˜70%successful-atbest(62-65%)¢ Essence:PatternRecognition¢ Key:EvolutionaryInformation Sequencehomologyimpliessimilarityinstructureandfunction Byinference/ByAnaysis¢ Databases(currently>500,000seq.,>17,000structuresInformationPrediction¢ Example:HomologousproteinsConservedCoreVariableLoopSecondaryStructurePredictionfor3-Model¢ Predict:α,β,loop,β-turn¢ Predict:membrane-spanning,α-helix¢ Predict:Amphipaticαβ¢ Predictionofthefoldedstructureoftryptophanesynthetaseandthecatalyticsubunitofc-AMPdependentproteinkinaseChou&Fossman(1974)¢ Frequencyofoccurrenceofagivena.a.inα.β.Loopinallproteinstructuresinthedatabase(statistical)¢ Nearestneighbors¢ ‡output:probabilityforeachresiduetobeinα.β.Loop¢ Artificialintelligence/neuralnetworks Trainacomputertorecognizepatternsthemoreinformationandthe“morepractice”thehighertheaccuracy(inprogress)Design¢ Minibody¢ Chymohelizyme¢ CalciumchannelMinibody¢ Synthetic(61a.a.)¢ Allβ¢ Template:heavychain,variabliedomainofIg¢ Hypervariableloops¢ Bindingsite:Histidinesineachhypervariableloop¢ Protein‡itfoldsandinbindsmetal(ZN+2)Chymohelizyme(Science248:1544,1990)¢ Design(computerbased):4helices,parallel,amphipathic,serineprotease¢ CatalyticTRIAD Ser,His,AspattheN-endofthebundleinthesamespatialarrangementaschymotrypsin¢ “oxyanionhole”andsubstratebindingpocketforacetyltyrosineethylester,aclassicalsubstrateofCTwereincludedinthedesign¢
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