紫外荧光在蛋白质分析中的应用

紫外与荧光光谱在蛋白质研究中的应用 主要内容 UV vis基础 实验要点 应用Fluorescence基础 实验要点 常规荧光 淬灭 荧光共振能量传递 FRET 谱学方法 10 100Kcal mol Bondsbreak Lightusedforvisionandphotosynthesis Electromagneticspectrum Nuclearspin vibrations Electronictransitions Thelongerthewavelength thelowertheenergy 基本原理1 LambertLaw Thefractionoflightabsorbedbyatransparentmediumisindependentoftheincidentintensity andeachsuccessivelayerofthemediumabsorbsanequalfractionofthelightpassingthroughit Log10 I0 I kl 基本原理2 Beer sLaw Theamountoflightabsorbedisproportionaltothenumberofmoleculesofthechromophorethroughwhichthelightpasses k c DeviationsfromtheBeer Lambertlaw 强吸收高浓度 噪声与误差 等吸收点 Isosbesticpoint 等吸收点常作为体系中只有两种成分 状态 的指示可作为参比波长若有等吸收点 则不需要为为每个谱作基线 单光路 双光路 双波长 二极管阵列检测器 二极管阵列光谱仪动力学实验 Wavelength Absorbance Concentration A B A B 波长选择 常规吸收光谱实验要点 样品准备 波长选择 池子选择 扫描速度选择 带宽选择 石英 玻璃 塑料 慢 快 若样品不稳定 若噪声大 分辨率低等 窄 宽 若噪声大 合适的浓度 正确的参比 紫外 可见光谱在蛋白质研究中的应用 浓度测定构象变化研究相互作用研究 蛋白质主链的紫外吸收 肽键在 250nm的远紫外区有较大吸收 蛋白质浓度测定 190nm 10000l mol cm 190nm比 280nm大 100倍但溶剂吸收也较大 溶剂的吸收 蛋白质中氨基酸的紫外吸收光谱 1mM 0 1mM 0 1mM 二硫键在250nm附近有弱吸收 确定蛋白质的消光系数 可以根据蛋白质序列预测蛋白质的消光系数ProtParam http cn expasy org tools protparam html Tyr的吸收谱与pH有关 pHtitrationcanbeusedtodetermine whetherTyrisinternalorexternal polarityofenvironment maxatpH6 274nm maxatpH13 295nm 环境极性的影响 BlueshiftofspectrainpolarsolventWeakerabsorptionofTyrinpolarsolvent 蛋白质构象变化研究 AbsorptionspectraofPoly L LysHCl randomcoilatpH6 0 25 C bold HelixatpH10 8 25 C dotted strandatpH10 8 52 C dashed 蛋白质折叠 变性研究 荧光光谱 0 3 3 0 TheFranck Condonprinciple transitionsareverticalinbothabsorptionandemission TheFranck Condonfactoristhesameforabsorptionandfluorescence 0 0 0 1 1 0 0 3 0 6 3 0 6 0 Exceptions verylonglivedS1state emissionoccursfromadifferentgeometry Reactionsfromtheexcitedstate 量子产率 F 发射的光子数 吸收的光子数 荧光强度 IF I0 1 10 cl F若 cl很小 则近似 IF I0 cl FInnerfiltereffect Innerfiltereffect 所以 实验中 A EX 0 1 RamanandRayleighScattering TwopotentialsourcesofbackgroundradiationareRamanandRayleighscattering Bothofthesephenomenaariseduetovibrationalchangesinducedinmoleculesbyincidentradiation Bothcanalsobedescribedasscatteredlight TheRamanlinesareofdifferentfrequencyfromtheincidentlightandusuallyoflongerwavelength Rayleighradiationisscatteredlightofthesamefrequencyastheincidentlight RayleighScattering 强度与r6 4成正比不能通过减空白来消除选择合适的激发波长从比激发波长大 10nm 处开始收谱减少带宽 Ramanscattering 因水中O H收缩 3300cm 1 1 RA 1 EX 0 00033 EnvironmentalSensitivity FluorophorescanbeaffectedbyalargenumberofenvironmentalfactorsincludingsuchparametersaspH ionicstrength non covalentinteractions lightintensity temperatureandsoon Boththeexcitationandemissionwavelengthsandthequantumyieldcanallbechangedbyenvironmentalfactors 温度 荧光一般随温度上升而减弱荧光实验需要恒温 2waysofmeasuringfluorescence Emissionspectrum excitation constant measurefluorescenceintensityofemissionagainst I e spectrumofemittedlight Excitationspectrum measurefluorescenceintensityatdifferentexcitation similartoabsorptionspectra Excitationspectrum Excitationspectrumshouldcorrespondcolselywiththeabsorptionspectrumofthemoleculethatisresponsibleforthefluorescence Excitationspectrumrevealswhetherthesampleishomogeneous andwhetherallfluorescencefeaturesresultfromasinglemolecule Experiments Spectralshifts Intrinsic ExtrinsicFluorescenceFluorescencequenching Internal ExternalFluorescenceResonanceEnergyTransfer FRET Rotationofmolecules fluorescenceanisotropyFluorescencelifetime Proteinintrinsicfluorescence Trpfluorescencecanbeselectivelyexcitedat295 305nm toavoidexcitationofTyr Trpisthedominantintrinsicfluorophoreinproteins Trpfluorescenceisverysensitivetoitslocalenvironment Itispossibletoseechangesinemissionspectrainresponsetoconformationalchanges subunitassociation substratebinding denaturation andanythingthataffectsthelocalenvironmentsurrondingtheindolering Also Trpappearstobeuniquelysensitivetocollisionalquenching eitherbyexternallyaddedquenchers orbynearbygroupsintheprotein Localelectricfieldscausespectralshifts Gasphase m m solvent SolventcanaffectthegroundstateandexcitedstatemoleculescausingspectralshiftExample Hbondingtotryptophan Changesitsabsorptionbyabout10nm Fluorescenceoftryptophandependsuponthedipolarnatureofthesolvent Ca parvalbumin tryptophanisburied 305nm Ca freeparvalbumin tryptophanisexposedtosolvent Tryptophaninvarioussolvents hexane trehaloseglass glycerol water Fluorescencespectralshiftscanbeverylarge Tyrfluorescence 若蛋白质只含TYR 不含TRP 蛋白质变性后 荧光强度明显增强 GFPisisolatedfromthePacificjellyfishAequoreavictoriaandnowplayscentralrolesinbiochemistryandcellbiologyduetoitswidespreaduseasaninvivoreporterofgeneexpression celllineage protein proteininteractionsandproteintrafficking GFPcanbefusedtoanotherproteineitherN orC terminally ThisisduetothefactthatbothterminiofGFPappearratherflexibleonthesurfaceofthebeta can sothatGFP sstructureisnotsignificantlydistortedordestroyedbythefusedprotein RibbondiagramoftheGreenFluorescentProtein GFP drawnfromthewild typecrystalstructure Theburiedchromophore whichisresponsibleforGFP sluminescence isshowninfullatomicdetail GreenFluorescentProtein GFP DifferentcolourformsofGFP AdditionallyseveraldifferentcolourformsofGFPhavebeenproduced blue cyan green andyellowFPorBFP CFP GFPandYFP InformationobtainedfromstudyingintrinsicfluorescenceThe3trpofahypotheticalproteinhaveamuchgreaterfluorescenceintensityandshorter maxthandoesfreetrp A withoutco factorB withcofactorC freetrp Whenthecofactorisadded intensitydecreasestheyhaveprobablybeenmovedtoamorepolar internalregion PropertiesoftheactivesiteofanenzymeAhypotheticalenzymeisknowntocontainasingletrpandits maxisnearlythesameasthatforfreetryptophansuggestingthatitisinapolarenvironment TheQislow suggestingthataquenchermightbenearby AcidtitrationdecreasesQandthepKofthetransitionisthatofacarboxylaasidechain 9 5 10 sothelowQiscausedbytheproximitytoacarboxyl Theadditionofthesubstrateshifts maxtoshorterwavelengths increasesQ Theshiftin maxindicatesthatthetrpisinalesspolarenvironment Thiscouldmeanthattherehasbeenaconformationalchangemovingthetrptoanon polarregionorthattrpisinornearthebindingsite thatsubstratebindingexcludeswater TheincreaseinQsuggeststhatcarboxylquenchingiseliminated Thelikelyansweristhatthetrpisintheactivesite thesubstratebindseithertothetrporthecarboxyl Fluorescenceemissionspectraofequalconcentrationsof1 8 ANSinethanol watermixtures Thelabelsadjacenttoeachcurveindicatethepercentageofethanolinthesolventmixture ProteinExtrinsicfluorescence ANS 1 anilinonaphthalene 8 sulfonicacid 1 苯胺基萘 8 磺酸 strongfluorescenceenhancementwhenitsexposuretowaterislowered Fluorescenceenhancementof1 8 ANS uponbindingtoprotein Theimageshowsaqueoussolutionsof1 8 ANSexcitedbyultravioletlight Additionofprotein bovineserumalbumin tothesolutioninthecuvetteontheleftresultsinintensebluefluorescence Thefluorescenceofuncomplexedfreedyeinthecuvetteontherightisnegligibleincomparison 1 8 ANSisasensitiveprobeforpartiallyfoldedintermediatesinprotein foldingpathways MoltenglobuleintermediatesarecharacterizedbyparticularlyhighANSfluorescenceintensitiesduetotheexposureofhydrophobiccoreregionsthatareinaccessibletothedyeinthenativestructure ANSbindingofsHSP16 5inGdnHCl TitrationofANSfluorescenceat475nm Haemoglobinisacomplexofasmallprostheticgroupwiththeproteinapohaemoglobin TheextrinsicfluorANSfluoresceswhenaddedtosolutionsofapohaemoglobinbutnotwithhaemoglobin Theadditionofhaemtotheapohaemoglobin ANScomplexeliminatesthefluorescencebydisplacingANS ThistellsusthatANSandthehaemgroupbindtothesamesite SinceANSisonlyfluorescentinhighlynon polarenvironments thehaemmustbindtoahighlynon polarsite ThiswouldgivevaluablecluesintheinterpretationofX raydiffractionpatterns E g youwouldknowthatacertainconfigurationofaminoacidscouldbethepointofinteractionbetweenhaemandproteinwhenbuildingthestructure Extrinsicfluorescenceexamples FluorescenceQuenching Internalquenchingduetointrinsicstructuralfeaturee g structuralrearrangement Externalquenchinginteractionoftheexcitedmoleculewithanothermoleculeinthesampleorabsorptionofexcitingoremittedlightbyanotherchromophoreinsample Intramolecularquenching TyroftenisquenchedbynearbytryptophanesTrpfluorescencecanbequenchedbyneighbouringprotonatedacidgroups IfthepKmeasuredbymonitoringtrpfluorescenceisthesameasthepKforaknownionisablegroup e g acarboxyl thenthegroupmustbenearthetrp Externalquenching Acrylamide I Cs Stern Volmerequation Fluorescencequenchingisafunctionof TheexcitedstatelifetimeThediffusion limitedquenchingconstantTheconcentrationofthequencher TheSternVolmerslope KSV skQ F0 F 1 tskQ Q Stern Volmerequation AsafirstproximationthevalueofKSVrevealsthedegreeOfexposuretothesolventofaTrpresidue 0M 1M 2M 3M 4M 5M 6M Stern VolmerplotatvariousGdnHClconcentrations AcrylamidequenchingofTrpfluorescence Stern Volmerconstants FluorescenceResonanceEnergyTransfer Knownasfluorescenceresonanceenergytransfer FRET orF rsterenergytransfer Itistheradiationlesstransferofexcitationenergyfromadonortoanacceptor Animportantconsequenceofthistransferisthatthereisnoemissionoflightbythedonor Theacceptormayormaynotbefluorescent FRETisadistance dependentinteractionwheretheenergytransferoccurstypicallyoveradistanceof1 10nm ThedistancedependentnatureofFRETishighlightedbythefactthatitisproportionaltotheinversesixthpoweroftheintermolecularseparation Ifthefluorophores extrinsicorintrinsic haveuniquelocationswithintheproteinorcomplex itispossibleforemissionlightenergyfromAtobeabsorbedbyBandtobeemittedaspartofB semissionspectrum Fluorescenceresonanceenergytransfer FRET FRETisdependentonthedistance R betweenthetwofluors Usedtomeasuredistancesinproteins membranes macromolecularassemblies10to80 apart Efficiencyoftheenergytransfer E fromdonortoacceptor AtoB definedbyE R06R06 R6 R distancebetweenA B R0isaconstantcalculatedfromabsorptionandemissionspectra Ecanbecalculatedfromfluorescenceintensity F E Fda FdFda Finthepresenceofacceptor Fd Fintheabsenceofacceptor OnceEisknown RcanbecalculatedfromthefirstequationifR0isknown FRETisusedasa spectroscopicruler theC terminalsubdomainofthemoleculeformsanensembleofcompactlooselyfoldedconformationswithnative likefeatures FRET应用实例 IntramolecularandintermolecularFRET a IntramolecularFRETcanoccurwhenboththedonorandacceptorchromophoresareonthesamehostmolecule whichundergoesatransition forexample between open and closed conformations IneachsquareboxcorrespondingtoCFPorYFP shownincyanoryellow respectively adiagonallinerepresentsthechromophore TheamountofFRETtransferredstronglydependsontherelativeorientationanddistancebetweenthedonorandacceptorchromophores theparallelorientationandtheshorterdistance 100 generallyyieldlargerFRET b IntermolecularFRETcanoccurbetweenonemolecule proteinA fusedtothedonor CFP andanothermolecule proteinB fusedtotheacceptor YFP Whenthetwoproteinsbindtoeachother FREToccurs Whentheydissociate FRETdiminishes FRETimagingmicroscopyexperiment InFRETexperiments asingletransfection intramolecularFRET orco transfection intermolecularFRET oftheconstructsmustfirstbeperformed TheoccurrenceofFRETcanbeobservedbyexcitingthesampleatthedonorexcitationwavelengthswhilemeasuringthefluorescenceintensitiesemittedatwavelengthscorrespondingtotheemissionpeaksofthedonorversusthoseoftheacceptor Iftheacceptoranddonorareatafavorabledistanceandorientation donoremissionintensitydecreases CFP cyan whiletheacceptoremission YFP yellow intensityincreases Advantages UsesofFRETCanprobethetopologyofmembraneproteinswhichcan tbedonebyX rayorNMR Quitefast simplecomparedto3 Dstructuredetermination Invivouses LimitationsLimitedrange 10 80 ifRis 8nmEisverysmallNeed2fluorophores adonor anacceptor AssumptionsaremadeinthecalculationofR0suchastheorientationofthedonor theacceptor 荧光光谱实验要点 样品纯度 浓度 A 0 1 恒温波长选择 Summary AllfluorescenceofaproteinisduetoTrp TyrorPhe unlessitcontainsanextrinsicfluororfluorescentco fact