《蛋白质晶体学》PPT课件.ppt

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 24http www rcsb org 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 Protein ProteinComplexExpressionandPurification 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 Protein ProteinComplexExpressionandPurification Protein DNAComplex 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 bindingproteinsoften ride ontheboundDNAandeluteatmoderateionicstrength DNAprecipitation e g viapolyethyleneimineaddition isauseful butsomewhatriskystep MostproteinsdonotbindtoSresinsatpH7 0 8 5 MajoritywillstillnotbindatpH6 0 7 0 thereforeanScolumnatpH6 0 8 0hasaverygoodpurificationfactoriftargetproteinisbound ACM column Optimizeproteinpurification hasanevenhigherpurificationfactor VirtuallynoproteinsbindtoCMcolumnsatpH 8 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 dryingorprolongedstorageat4 C Storagesomeproteinsin30 50 glycerolorethyleneglycolat 20 Cor 80 Cisausefulalternative 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 Crystallizationofamacromo