分子生物学蛋白质结构与功能the structure of proteins

TheProteinsXiangweiGao(高向伟)xiangweigao@

BioelectromagneticsLabotary,ZhejiangUniversity,SchoolofMedicneResearchBuildingB203WhyDoWeStudyProteins?ProteinsarecodedbygenesTheyplaycrucialfunctionalrolesinalmosteverybiologicalprocessTheLifeCycleofAProtein翻译成熟糖基功能调节磷酸化执行功能结构功能降解Wheredoesaproteincomefrom?Howisaproteinprocessed,translocatedtotheproperplaceanddegraded?Howtodescribethestructureofaprotein?Whatarethefunctions?Howtostudytheprotein(s)?Proteinsynthesis(Translation)蛋白质翻译Proteinmaturation(folding,modification),anddegradation蛋白质成熟、降解Structureandfunctionofprotein蛋白质的结构与功能Methods:protein-proteininteractionetal蛋白-蛋白相互作用TheStructureofProteinsOutlineAminoacids:propertiesProteinstructure:primary,secondary,tertiary,andquaternarystructuresmethodsforproteinseparationandpurificationmethodstostudyproteinstructuresAminoAcidsandPeptidesAminoAcids(氨基酸):BuildingblocksThereareover300naturallyaminoacidsonearthOnly20aminoacidsparticipateinproteinsformationRNonpolar(hydrophobic)非极性Polar(hydrophilic)极性中性Acidic酸性Basic碱性ClassificationsofAminoAcidsNonpolarhydrophobicAAsHCHNH3COOH3CCHNH3COOCHCHNH3COOH3CH3CCH2CHNH3COOCHH3CH3CCHCHNH3COOCH2H3CH3CGlycine(Gly)Isoleucine

(Ile)Leucine

(Leu)Valine

(Val)Alanine

(Ala)甘氨酸丙氨酸缬氨酸亮氨酸异亮氨酸CH2CHNH3COO苯丙氨酸Phenylalanine

(Phe)CHCOOH2NH2CCH2CH2脯氨酸Proline

(Pro)GAVLIFPMMethionine

(Met)CH2CHNH3COOCH3SCH2甲硫氨酸WTryptophan

(色氨酸CH2CHNH3COONHCHCTrp)2.PolarunchargedAAsSNYQCTCysteine

(Cys)Serine

(Ser)丝氨酸酪氨酸半胱氨酸天冬酰胺Asparagine

(Asn)谷氨酰胺Glutamine

(Gln)CH2CHNH3COOHOCH2CHNH3COOHSCH2CHNH3COOTyrosine

(Tyr)HOCH2CHNH3COOCCHCHNH3COOH3CCH2CHNH3COOH2NOCH2CH2NOHO

苏氨酸Threonine

(Thr)3.AcidicAAs4.BasicAAsDEKRH

SpecialAAsGlycinesidechainis–H,veryflexibleProlinehastwocovalentbondswithbackboneCysteinecanformdisfulfidebridgePropertiesofAminoAcidsAminoacidshavebothacidandbaseproperties(两性解离)Aromaticaminoacidsabsorblightinthenear-ultraviolet(近紫外吸收)Colouredbyninhydrin(茚三酮反应)EdmanreactionAminoAcidsHaveBothAcidandBaseProperties(两性解离)pH=pIpH<pIpH>pIpI,isoelectricpoint,等电点:TheisoelectricpointisthepHatwhichaparticularmoleculeorsurfacecarriesnonetelectricalchargeAromaticAminoAcidsAbsorbLightintheNear-Ultraviolet(近紫外吸收)Aromaticaminoacids(芳香族氨基酸):Trp,Tyr,PheNear-ultravioletCanbeusedforproteinquantitationNinhydrinReaction(茚三酮反应)440nmforproline,hydroxyproline540nmforotherAAs黄色紫色离子交换层析柱茚三酮EdmanReaction(PITC,苯异硫氰酸酯)偶联反应环化断裂反应转化反应Peptides(肽)Peptidesareshortpolypeptideschains,uptolengthofabout50aminoacids.Proteinsaremadeupofoneormorepolypeptideswithmorethan50aminoacids.Theatomsofthegroup,O=C-N-H,togetherwiththetwoC,arefixedonthesameplane,knownasthepeptideplane.

ThewholeplanemayrotatearoundtheN-Cbond(phiangle)orC-Cbond(psiangle).ThePeptidePlane肽平面ApeptideplaneNativeFunctionalPeptides谷胱甘肽(glutathione,GSH)TheMolecularStructureofProteinHowtoDescribeProteinStructure：

Primary:AminoacidsequenceSecondary:Alphahelix,betasheetandloopsTertiary:Thethree-dimensional(3D)structureQuaternary:Arrangementofseveralpolypeptide

chainsTheorderofaminoacidsinaproteinisgeneticallydeterminedContainsalltheinformationtoassumeitscorrect3-DstructurePrimaryStructure-helix(-螺旋)-sheet(-折叠)-turn(-转角)Secondarystructure-helix-helixisthemostpopularsecondarystructureelementsinproteinsmostofthe-helicesareright-handedEverybackboneN-HgroupformesahydrogenbondtothebackboneC=OgroupoftheaminoacidfourresiduesearlierEachhelixcontains3.6AAsThepitchis0.54nm-sheetItturnsforabout180oItcontains4AAs.ThefirstandtheforthaminoacidformH-bond-turnMotif(模序)Themotifisacharacteristicdomainstructureconsistingoftwoormoresecondarystructures,whichappearsinavarietyofproteins.Examples:helix-loop-helix,leucinezipper,zincfinger,etcHelix-loop-helix螺旋-环-螺旋Two-helicesconnectedbyaloopThesmallerhelicesformdimersThelargerhelixcontainstheDNA-bindingregionsAcommonmotifintranscriptionfactors:c-mycLeucineZipper亮氨酸拉链ParallelalphahelicesAleucineresidueateveryseventhpositionineachhelixOnebasicregionforDNAbindingAcommonmotifintranscriptionfactors:c-jun,c-fosZincFinger锌指Twoantiparallel-strandsandan-helixConsensussequence:Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-HisManytranscriptionfactors,regulatoryproteins,andotherproteinsthatinteractwithDNAcontainzincfingers-helixofeachfingercontactstheDNAThedifferentsectionsof-helix,-sheet,otherminorsecondarystructureandconnectingloopsofapolypeptidefoldinthreedimensionsTertiaryStructureShape-DeterminingInteractionsinProteinsNoncovalentinteraction(非共价结合)

hydrogenbonds氢键N-H…OvanderWaalsforces范德华力

hydrophobicinteractions疏水作用

electrostaticsaltbridges离子键Covalentinteraction(共价结合)disulfidebonds二硫键ProteinDomains(结构域)Aproteindomainisapartofproteinsequenceandstructurethatcanevolve,function,andexistindependentlyoftherestoftheproteinchain

结构上及功能上BuiltfromdifferentcombinationsofsecondarystructureelementsandmotifsQuaternaryStructureArrangementofseveralpolypeptidechainsEachpolypeptidechainiscalledsubunit(亚基)HydrogenbondsandelectrostaticsaltbridgesaremajorforcesItallowsverylargeproteinmoleculestobemade,suchascollagen(大分子形成)Itcanprovidegreaterfunctionalitytoaproteinbycombiningdifferentactivitiesintoasingleentity(具有多种功能)Theinteractionsbetweenthesubunitscanoftenbemodifiedbybindingofsmallmoleculesandleadtotheallostericeffects(变构效应)AdvantagesofthequaternarystructureQuaternaryStructureofHemoglobin(血红蛋白)ProteinStructureStudyContentsSeparation&PurificationAllmethodsarebasedonthepropertiesoftheinterestedproteinsPrimarystructureanalysisSequencing,Massspectrometry(质谱)TertiarystructureanalysisX-raycrystallography(X-线晶体衍射),NMR(核磁共振),BioinformaticsPartI:Separation&PurificationTheprincipalpropertiesofproteinsusedforseparationandpurificationSizeDialysis透析,Ultrafiltration超滤,Gelfiltrationchromatography凝胶过滤层析,Densitygradientcentrifugation密度梯度离心ChargeElectrophoresis电泳,Isoelectricfocusing等电聚焦,Ion-exchangechromatography离子交换层析Hydrophobicity疏水作用

Hydrophobicinteractionchromatography疏水作用层析Affinity亲和作用

His,GST,HA,FLAGtagsDialysis(透析)&Ultrafiltration(超滤)用途：蛋白质去盐、浓缩DensityGradientCentrifugation(密度梯度离心)根据蛋白质大小进行分离GelFiltrationChromatography(凝胶过滤层析)+SDS-PolyAcrylamideGelElectrophoresisSDS(聚丙烯酰胺凝胶电泳)SDS:maketheproteinchargedSmallmolecule——fastBigmolecule——slow4321MkDa1,antiserum;2,pelletafter50%(NH4)2SO4precipitation3,supernanantafter50%(NH4)2SO4precipitation4,crudepurifiedpolyclonalantibodyafter30%(NH4)2SO4precipitationSDSof(NH4)2SO4precipitatedantibodiesHeavychainLightchain4726IsoelectricFocusing(等电聚焦)2-DimentionalElectrophoresis(2-D)

(双相电泳)Ion-ExchangeChromatography(盐离子交换层析)Enzyme-substrate

bindingGST(谷胱甘肽转移酶)Biotin-Strepavidin,[His]6-Ni+Receptor-ligandbindingAntibody-antigenbindingHA-tag&antibodyMyc-tag&antibodyAffinityChromatography(亲和层析)PartII:PrimarystructureanalysisSequencingMassspectrometryWorkFlowforProteinSequencingBreakanydisulfidebridgesintheprotein.(破坏二硫键)Separateandpurifytheindividualchainsoftheproteincomplex,iftherearemorethanone.(分离多肽链)DeterminetheAAcompositionofeachchain.(确定氨基酸组成)Determinetheterminalaminoacidsofeachchain.(确定N端、C端)Breakeachchainintofragmentsunder50aminoacidslong.(降解)Separateandpurifythefragments.(分离纯化每个肽段)Determinethesequenceofeachfragment.(确定每个肽段序列,Edman反应)Repeatwithadifferentpatternofcleavage.(选择令一种降解方法，重复步骤5-7)Constructthesequenceoftheoverallprotein.(重叠确定序列)From:Wikipedia,thefreeencyclopediaExample:SequencingofInsulinFrederickSangerNobelPrizeinChemistry1958MassSpectrometry(MS)质谱Massspectrometryisananalyticaltechniquethatidentifiesthechemicalcompositionofasamplebasedonthemass-to-chargeratioofchargedparticles.Ionsourcetechnologiesmatrix-assistedlaserdesorption/ionization(MALDI)基质辅助激光电离

electrosprayionization(ESI)电喷射离子化Massanalyzertechnologies

Time-of-flight(TOF)DatapresentationIdentifytheAminoAcidSequenceofProteinwithMS/MSMS/MS+++++1peptideselectedforMS/MSThemassesofallthepiecesgiveanMS/MSspectrumPeptidemixture+++++UsethefragmentionmassesasspecificpiecesofthepuzzletohelppiecetheintactmoleculebacktogetherPartIII:TertiaryStructureAnalysisX-rayCrystallographyNuclearMagneticResonance(NMR)Cryo-electronmicroscopy(CryEM)BioinformaticsOthermethodsX-raycrystallography(X-线晶体衍射)X-raycrystallographyisamethodofdeterminingthearrangementofatomswithinacrystal,inwhichabeamofX-raysstrikesacrystalandscattersintomanydifferentdirections.Fromtheanglesandintensitiesofthesescatteredbeams,acrystallographercanproduceathree-dimensionalpictureofthedensityofelectronswithinthecrystal.Fromthiselectrondensitymap,themeanpositionsoftheatomsinthecrystalcanbedetermined,aswellastheirchemicalbonds,theirdisorderandsundryotherinformation.Themostaccuratemethodforprotein3Dstructuredetermination.Crystalstructureonly,staticstructure.X-rayCrystallographyProcess1,000,000x100,000,000X1,000XLightmicroscope(λ=500nm)ElectronmicroscopeX-rayCrystallography(λ=0.154nm)TheResolutionDependsontheWavelenghWhathappenstoelectronwhenitishitbyX-rays?Theelectronstartsvibratingwiththesamefrequencyasthex-raybeam.Asaresult,secondarybeamswillbescatteredinalldirections.PrimarybeamSecondarybeamsScatteringF