《蛋白质晶体学》PPT课件

ProteinCrystallography,汪德强,重庆医科大学医学检验系生物技术教研室感染性疾病分子生物学教育部重点实验室,一、概述1历史的回顾1895年德国物理学家伦琴发现X射线并因此获得1901年首届诺贝尔物理学奖，X射线历经110年跨越3个世纪，由于众多学者在探索X射线性质、应用、仪器等方面的创新性研究，先后有29位物理学家、晶体学家、化学家、分子生物学家等分别获得了物理（7项）、化学（9项）、生理学或医学（3项）总计19项诺贝尔奖。,1912年劳厄获得了X射线通过晶体后产生的衍射斑点图像（劳厄衍射图），证明了X射线的波动性及其波长范围。随后提出了表示原子排列周期与X射线波长间关系的著名的衍射方程（劳厄方程），并成功地解释了晶体衍射的实验结果。英国物理学家布拉格父子、达尔文等人发展了X射线衍射理论，类比光学反射原理提出了表示晶体结构（晶面间距d）、X射线波长（）与衍射方位（）间的关系的布拉格方程，提出了嵌镶晶体、完整晶体和包含有原子热运动诸因素的衍射强度公式，阐明了X射线,通过晶体产生衍射的付里叶变换本质，获得了X射线的连续光谱与取决于阴极材料的特征光谱。康普顿发现了X射线二次散射时引发的波长的变化(康普顿-吴有训散射)而确定了其粒子性质，从而揭示了X射线的波动与粒子二象性。之后，全世界众多的物理实验室相继开展了对X射线的基础研究工作，并逐步拓展为一个多学科交叉研究热点，主要的应用领域包括：矿物学、物理学、有机与无机化学、分子生物学、医药学、金属与材料科学等。,并最终使X射线衍射成为有机分子（特别是生物活性分子）立体结构测定的有力工具，为研究生理活性物质（药物分子）的立体结构、结构改造、结构预测、结构功能关系为目标的有机晶体学科奠定了基础。对于生物大分子的研究，始于30年代中期，贝纳尔和藿奇金开始用X射线衍射方法研究胃蛋白酶的晶体结构，但直到布拉格主持凯文迪实验室后，才使得这一工作取得突破，为创建分子生物学科奠定了基础。1953年沃森和克里克根据X衍射实验数据建立了脱氧核糖核酸(DNA)的双螺旋结构，并因此获得1962年的诺贝尔生理学和医学奖。,肯德鲁和佩卢茨从30年代开始，应用X衍射方法研究肌红蛋白与血红蛋白的晶体结构，历经20多年的艰苦努力，在众多科学家的共同参与下，终于在1960年获得了这两个蛋白质的三维结构，并因此荣获1962年的诺贝尔化学奖。在1957至1967年的10年中，相继用X衍射方法测定了溶菌酶、胰岛素、胰凝乳蛋白酶A、核糖核酸酶、核糖核酸酶S和羧肽酶的高分辨晶体结构。戴森豪菲尔和胡贝尔、米海尔因测定紫色细菌光合作用中心的三维结构而获得1988年的诺贝尔化学奖，形成了新的蛋白质晶体学科与结构分子生物学科。,物理奖（7项8人）,化学奖（9项15人）,生理医学奖（3项6人）,相关学科发展（2项2人）,2X射线晶体结构分析,X射线：表示所用的物理源与晶体相互作用的物理效应衍射晶体：表示固体状态下的一种特殊存在形态晶体生长晶体的几何性质对称性衍射信息中的对称性相位计算中的对称性结构分析：两次付里叶变换，完成第二次付里叶变换的数学方法晶体结构描述,LicTmutant(active)H207D/H269D,LicTwt(inactive),ComparisonoflicT-wtandlicTmutant,Graille*andZhou*etal.2004vanTilbeurghetal.EMBOJ.2001,Yangetal.EMBOJ.2002,Structure-directeddrugdesign,AnexampleofThy1fromThermotogamaritimaThy1:thymidylatesynthase-complementingproteinpresentinarchaea,prokaryotes,virusesNOTineukaryotes,Lesley,SAetal.PNAS;2002,Thy1-FAD-dUMP,Thy1-dUMP-HEPES,PDBContentGrowth(2004/08/01),OutputfromInternationalStructuralGenomicsConsortiaContributionfromcrystallographers,2004/04/13,OutputfromInternationalStructuralGenomicsConsortiiumContributionfromNMRspectrometrists,2004/04/13,FutureorientationsofSG1,Reconstructionofmultiproteincomplexes(basedoninteractomics)2,Systematicallysolvingthe3-Dstructuresofmembraneproteins(achallengeofnoveltechniques)3,SystemsBiology,Interactomes:1,Yeasttwo-hybrid2,TAP(tandemaffinitypurification)3,MassSpectrometry4,Co-IP(coimmunoprecipitation)5,Phagedisplay,OverexpresstheputativeproteincomplexinvivoorReconstructitinvitrofromtheindividualproteins,Solvethe3-DstructurebymeansofX-raycrystallographyCryo-ElectronMicroscopyElectroncrystallography(2DEM)Electrontomography,SystematicallyStructuretheMembraneProteins:Abigchallenge!PDB:26,880structures,updatedon2004/08/24/pdb/index.htmlMembraneproteins:81structures,updatedon2004/06/15http:/www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html,StructuralBiologyProcesses,X射线衍射实验和结构计算过程Fourier变换与Fourier反变换,Geneofinterest,Idealcase,Tragicreality,Designmultipleconstructs,Studyliteratureandanalog/modelcases,Evaluateandoptimizeexpression,Small-scalepurification,Evaluateproteinquality,Large-scalepurification,Screening,Selectexpressionsystem(s),Onlyafew(orone)constructs,Newproteinwithlittlepriorknowledge,Sub-optimalexpression,Purification,Limitedchoiceofexpressionsystems,I.Recombinantproteinover-expressionandpurification,Expressionsystems:BacteriasystemYeastInsectcellsMammaliancellsCell-freesystem,SomeVectorsforE.coliExpressionSystem,ProteinExpressioninYeast,Cloningoftargetgenetovector,TransformtoyeastPichiapastoris,Selectionofrecombinantyeaststrain,Yeastcellcultureforproteinproduction,ProteinExpressioninInsectCells,Afterrecombination,CloningoftargetgenetopFastBac,TransformtobacteriawithBacmid,Bacmidtransfectedtoinsectcells,Virusassemblyininsectcells,VirusesinfectInsectCellsforproteinproduction,Strainsforexpression:Sf9,Sf21,Hi5,TransientExpressionInMammalianCells,293Ecellcanbeculturedinsuspensionmedium,Recombinantplasmidwithtargetgene,Transfectto293EcellswithPEI,Harvestcellsforproteinpurification,293EBNA1CellsWithGFPExpressingVector,A,B,Wholecellsonplate;CellsinthesameplatetoAviewedbyGFPflorescence,RecombinantProteinsExpressionIn293EBNA1Cells,Lanes:1.Proteinstandard;2.Controlwhole293Ecells;3.GFPexpressed293Ecells;4.HCF-1N380expressed293Ecells;5.HCF-1N16-363expressed293Ecells.,Recombinantprotein1(lane4),12345,14,20,31,45,67,94,Recombinantprotein2(lane5),GFP(lane3),Cell-freeSystemforProteinProduction,Sometimesitcanproducesolubleproteinwhichcannotbeexpressedassolubleformwithcellularsystem.,Roche:RapidTranslationSystem(RTS),Rapidproteinexpression,Toxicproteinexpression,ProteinProteinComplexExpressionandPurification:a.Proteinsexpressseparately;b.Proteinsco-expressinonecell.2.Protein-NucleicAcid：a.Protein-DNAComplex；b.Protein-RNAComplex.,ProducingProteinComplexesforCrystallization,MethodsforproductionofrecombinantproteincomplexesbyinvivoreconstitutioninE.coli1.Usecompatiblevectors,suchaspMR101(p15Aori)andpET15B(pBR322ori);2.Useonevectorwithmorethanoneexpressioncassettes-polycistronic；Benefitsofinvivoreconstitution(coexpression)efficiencyoneroundofexpressiononeroundofpurificationqualitycoexpressionandcofoldingofpolypeptidesinthepresenceofcellularchaperonesmayincreaseyieldoffunctionalcomplex,ProteinProteinComplexExpressionandPurification,ProteinDNAComplex,Proteinsolubility:higherinhighsaltbufferusually;Protein-DNAcomplexstability:morestablethanproteinalone;DNAlengthandsequenceusedforcrystallization:a.additionalbasepairs;b.stickyends;4.PurificationofDNAoligos:HPLCwithhydrophobicinteraction,C4etc;5.Trappingreactionintermediate:disulfidebridge;proteinpointmutation,etc;6.Preparationofprotein-DNAcomplexes:mixwithextramolarDNA;Crystallization:PEGorMPDinlowslatbuffer;Example:over6000trialforprotein-DNAcomplex.,Protein-RNAComplex,Difficulties:avoidofRNase!1.Phosphategroupsinterferecrystalpacking;2.ElongatedRNAspackloosely;RNAengineering:bluntorstickyends;deletion,replacement,etc;RNApreparation:1.Synthesis;2.Invitrotranscription;,ProteinModificationforCrystallization,1.Proteininhibitor,partnerandmonoclonalantibody;2.Proteinpost-translationalmodification;3.Proteinmutagenesis:truncation,mutation,deletion,ProteinMutagenesis,1.Truncationordeletion:secondarystructureprediction;DXMSresult;homologueproteinsequencescomparisonorstructurecomparison;Mutationmethodsa.Selectedpointmutation;b.Randommutation：DNAshufflingforchimericprotein;randommutationbylow-fidelityPCR.,Hydrogen/deuteriumexchangemassspectroscopy(DXMS)forproteinanalysis,Keenan,RobertJ.etal.(2005)Proc.Natl.Acad.Sci.USA102,8887-8892,RandommutationbyDNAshuffling,MutationselectionbyGFPfoldingreporter,GFP,TargetProtein,CorrectFoldingofTargetProtein,MisfoldingofTargetProtein,Fluroscence,NH3+,COO-,NoFluroscence,(Waldo,GS.Etal.1999,NatureBiotech.17:691),GFPFoldingReporter,Wild-typegene,RandommutagenesiswithPolymerase(Exo-),RandomMutagenesis.CloneintoGFPvector.Selectthebrightestcolonies.TestthesolubilityofKelch-GFP.RecloneintoGSTfusionvector.TestthesolubilityofGST-Kelch.,PCR,Proteinpurificationmethod,AffinityColumn:bytagsorantibodies;Ionexchangecolumn;Sizeexclusioncolumn;Hydrophobicinteraction;others,MetalaffinityorotheraffinitycolumnsTCEPisaverygoodalternativetoDTTorBMEwhenyoumusthaveareducingagentduringpurification.MostproteinswillbindtoQresinsatpH7.0-8.5.CheckifDEAEcanbeusedsinceitspurificationfactorismuchhigher.LowerpHresultsinhigherpurificationfactoraslongastargetproteinstillbinds.DNA-bindingproteinsoftenrideontheboundDNAandeluteatmoderateionicstrength.DNAprecipitation(e.g.viapolyethyleneimineaddition)isauseful,butsomewhatriskystep.MostproteinsdonotbindtoSresinsatpH7.0-8.5.MajoritywillstillnotbindatpH6.0-7.0,thereforeanScolumnatpH6.0-8.0hasaverygoodpurificationfactoriftargetproteinisbound.ACM-column,Optimizeproteinpurification,hasanevenhigherpurificationfactor.VirtuallynoproteinsbindtoCMcolumnsatpH8.0.TheuseofacidiccolumnsmayrequirepassingthroughthepIoftargetprotein.Hydroxyapatitecangiveveryhighpurificationfactors.Size-exclusionchromatographyisveryusefulandnormallynon-damagingmethod.Purificationbyproteinproperties,Optimizegeneorexpression,Apparentproblem,Misfolding,Lowrateofsynthesis,Proteindegradation,Expressionsystem,Fusionsortags,Promoters,Expressionconditions,Codonbias,Co-expression,Domainstructure,Possiblechanges,Misfolding,FoldingefficiencyLackofproperchaperones.SynthesisrateSynthesisistoofastforthefoldingcapacityofthesystem.ProteinlocalizationProteinrequiresspecificcompartmentalization(i.e.periplasmicorintramembrane)tofold.Post-translationalmodificationEukaryoticproteinsoftenrequirespecificPTMtomature.,FoldingefficiencyToxicproteinsareoftendominant-negative.Asaresult,theworseisthefoldingofsuchproteins,themore(incompetent)proteinisactuallymade.SynthesisrateSynthesiscanbenegativelyaffectedbyinitiationrate,codonbias,nopropernutrientsorlow-levelco-factors(e.g.certainmetalions).ProteinlocalizationProteinistranslocateddirectlyintoaspecificcompartment(i.e.periplasmicorintramembrane).Asaresult,ifthecompartmentisnotavailable,theribosomesstallorabort.,Lowrateofsynthesis,FoldingefficiencyIfinclusionbodiesarenotformed,improperlyfoldedproteincanberapidlydegraded.SynthesisrateLowrateofsynthesiscanresultintheneedforlongergrowthtimesandthereforelongerexposureoftheproteintoproteases.ProteinlocalizationProteincompartmentalizationcanhavesignificanteffectondegradation,e.g.whenproteinissubjectedtosignalpeptidasesinbacterialperiplasm.Post-translationalmodificationEukaryoticsystemsuseubiquitinylationasdegradationsignal.Membrane-associatedproteasescanspecificallyattackproteinsthatbearmembrane-associationortransmembranesignals.,Proteindegradation,FusionsortagsCanhaveatremendousnegativeorpositiveeffectonfoldingCo-expressionCanbeveryhelpfulExpressionconditionsLoweringthetemperatureoftenresultsinmorefoldedprotein.Functionalexpressioncanalsoberegulatedthroughnutrientsandco-factors.DomainstructureProperdefinitionofdomainboundariescanhaveparamounteffectonfolding.,Expressionsystem,Fusionsortags,Expressionconditions,Foldingefficiency,Co-expression,Domainstructure,ExpressionsystemItiseasier(andcheaper)toproducemassivequantitiesofproteinsinbacteriaoryeast.PromotersExpressionconditionsTemperature,nutrient/oxygencontent,antibiotics,etc.DomainstructureTranslationalinterdomainpausingcanslowdowntheoverallprocessorresultinabortiveexpression.CodonbiasCodonoptimizationensuresthatrarecodonsdonotcausetranslationalpausingorabortion.,Expressionsystem,Fusionsortags,Expressionconditions,Synthesisrate,Promoters,Domainstructure,Codonbias,Avoidfreeze-thawcycles.Mostproteinsdonottoleratefreeze-dryingorprolongedstorageat4C.Storagesomeproteinsin30-50%glycerolorethyleneglycolat20Cor80Cisausefulalternative.Flash-freezingproteinstockinsmallaliquots.,Optimizeexistingsampleproperties,II.ProteinCrystallization,Generalapproachforproteincrystallization,Macromolecularcrystalsarecomposedofapproximately50%solventonaverage,thoughthismayvaryfrom25to90%dependingontheparticularmacromolecule.Macromolecularcrystalgrowthisstilllargelyempiricalinnature.Itisstillamysteryforthereasonsthatsomeproteinscouldnotbecrystallized.Searchingsystematicallyandbroadly;,Crystalscreening,Crystaloptimization,Twostepsforproteincrystalobtaining,Screening,Robotic,Manual,CheapTime-testedReadilyavailableAllowsforcreativity,MultitudeofconditionsHighlyreproducibleEasytodocumentandtrackdataLowerconsumptionofprotein,1.Alteringtheproteinitself:suchaschangeofpHtoalterproteinionicsurface;2.Byalteringthechemicalactivityofthewater:e.g.,byadditionofsalt;3.Byalteringthedegreeofattractionofoneproteinmoleculeforanother:e.g.,changeofpH,additionofbridgingions;4.Alteringthenatureoftheinteractionsbetweentheproteinmoleculesandthesolvent:e.g.,additionofpolymersorions.,Crystallizationofamacromol