CircularDichroism-(1)

圆二色谱CircularDichroism(CD)Application圆二色光谱仪通过测量生物大分子的圆二色光谱从而得到生物大分子的二级结构。可应用于：蛋白质折叠﹑蛋白质构象研究,DNA/RNA反应,酶动力学,光学活性物质纯度测量,药物定量分析。天然有机化学与立体有机化学,物理化学,生物化学与宏观大分子,金属络合物,聚合物化学等相关的科学研究。构象确定蛋白质构象最准确的方法是x-射线晶体衍射，但对结构复杂、柔性的生物大分子蛋白质来说，得到所需的晶体结构较为困难。二维、多维核磁共振技术能测出溶液状态下较小蛋白质的构象，可是对分子量较大的蛋白质的计算处理非常复杂。圆二色光谱：研究稀溶液中蛋白质构象，快速、简单、较准确CDisveryusefulforlookingatmembraneproteinsMembraneproteinsaredifficulttostudy.Crystallographydifficult-needtousedetergentsOftenevenwhenstructureobtained:Q-isitthesameaslipid?CDidealcandospectraofproteininlipidvesicles.WewilllookatStaphylococcala-hemolysinasanexample主要内容CD原理蛋白质CD谱CD实验要点CD原理圆二色性(circulardichroism,CD)当平面偏振光通过具有旋光活性的介质时，由于介质中同一种旋光活性分子存在手性不同的两种构型，故它们对平面偏振光所分解成的右旋和左旋圆偏振光吸收不同，从而产生圆二色性．圆二色性的表示椭圆度，摩尔椭圆度[]=2.303(AL–AR)/4[]=3298(L-R)3300(L-R)在蛋白质研究中，常用平均残基摩尔椭圆度圆二色仪原理蛋白质的CD谱Thepeptidebondisinherentlyasymmetric&isalwaysopticallyactive蛋白质的光学活性蛋白质的CD谱CDspectrainthefarUVregion(180nm–250nm)probesthesecondarystructuresofproteins.CDspectrainthenearUVregion(~250and~350)monitorsthesidechaintertiarystructuresofproteins.NearUVCDspectrum蛋白质中芳香氨基酸残基，如色氨酸(Trp)、酪氨酸(Tyr)、苯丙氨酸(Phe)及二硫键处于不对称微环境时，在近紫外区250～320nm，表现出CD信号。Phe残基:255、261和268nm附近；Tyr残基:277nm左右；而在279、284和291nm是Trp残基的信息；二硫键的变化信息反映在整个近紫外CD谱上。近紫外CD谱可作为一种灵敏的光谱探针，反映Trp、Tyr和Phe及二硫键所处微环境的扰动，能用来研究蛋白质三级结构精细变化。NearUVCDspectrumofLysozyme260270280290300310-200-1000100nmMainCDfeaturesofprotein2ndarystructures-band(nm)+band(nm)α-helix222208192β-sheet216195β-turn220-230(weak)180-190(strong)205polyproIIhelix190210-230weakRandomcoil200212FarUVCDspectraofpoly-L-LysCDsignalsforsamesecondarystructurecanvary(abit)withenvironmentLau,TanejaandHodges(1984)J.Biol.Chem.259:13253-13261Effectof50%TFEonacoiled-coilwavelengthinnm200210220230240MRE-35-30-25-20-15-10-50TM-36aqueousTM-36+TFETFEButonacoiled-coilbreaksdownhelicaldimertosinglehelicesAlthough2ndrystructuresameCDchangesEffectof50%TFEonamonomericpeptidewavelengthinnm200210220230240MRE-35-30-25-20-15-10-50peptideinwaterpeptidein50%TFETFECanseethisbylookingattheeffectoftrifluoroethanol(TFE)onacoiled-coilsimilartoGCN4-p1TFEinduceshelicityinallpeptidesBestfittingproceduresusemanydifferentproteinsforstandardspectraTherearemanydifferentalgorithms.Allrelyonusingupto20CDspectraofproteinsofknownstructure.Bymixingthesetogetherafitspectraisobtainedforanunknown.ForfulldetailsseeDichroweb:theonlineCDanalysistoolCangenerallygetaccuraciesof0.97forhelices,0.75forbetasheet,0.50forturns,and0.89forotherstructuretypes(Manavalan&Johnson,1987,Anal.Biochem.167,76-85).估算蛋白质a螺旋含量仅适合a含量较高的蛋白质！*Yang算法LimitationsofCDsecondarystructureanalysisThesimpledeconvolutionofaCDspectruminto4or5componentswhichdonotvaryfromoneproteintoanotherisagrossover-simplification.ThereferenceCDspectracorrespondingto100%helix,sheet,turnetcarenotdirectlyapplicabletoproteinswhichcontainshortsectionsofthevariousstructurese.g.TheCDofanα-helixisknowntoincreasewithincreasinghelixlength,CDofβ-sheetsareverysensitivetoenvironment&geometry.FarUVcurves(275nm)cancontaincontributionsfromaromaticamino-acids,inpracticeCDismeasuredatwavelengthsbelowthis.TheshapesoffarUVCDcurvesdependontertiaryaswellassecondarystructure.蛋白的三级结构1976年，Levitt和Chothia曾在Nature上报道，规则蛋白质的三级结构模型可分为4类(1)全α型，以仅α-螺旋结构为主，其分量大于40％，而β-折叠的分量小于5％(2)全β型，以β-折叠这种结构为主，其分量大于40％，而仅一螺旋的分量小于5％；(3)α+β型，α螺旋及β-叠折分量都大于15％，这两种结构在空间上是分离的，且超过60％的折叠链是反平行排列；(4)α/β型，α-螺旋和B-折叠含量都大于15％，它们在空间上是相间的，且超过60％的折叠链平行排列。CDsignalofaproteindependsonits2ndarystructure——chymotrypsin(allb)——lysozyme(a+b)——triosephosphateisomerase(a/b)——myoglobin(alla)从CD谱分析蛋白质的结构类型(Venyaminov&Vassilenko)DEF_CLAS.EXE:对全a、a/b和变性蛋白质的准确度为100%，对a+b的准确度为85%，对全b的准确度为75%。对多肽的判断较差！CD实验要点DeterminationofProteinConcentration精确的方法有：1定量氨基酸分析；2用缩二脲方法测量多肽骨架浓度或测氮元素的浓度；3在完全变性条件下测芳香氨基酸残基的吸收，来确定蛋白质的准确浓度.NotAcceptable:1.BradfordMethod.2.LowryMethod.3.Absorbanceat280and/or260nm.NitrogenflushingFlushingtheopticswithdrynitrogenisamust:Xelamphasaquartzenvelope,soifoperatedinairit’lldevelopalotofozone,harmfulforthemirrorsbelow195nmoxygenwillabsorbradiationHTplotTheHTplotisveryimportant,sincereadingsabove600-650Vmeanthatnotenoughlightisreachingthedetectorsoasampledilutionortheuseofshorterpathcellarerequired.FurthermoretheHTplotisinrealtyasinglebeamspectraofoursample,sincethereisadirectrelationbetweenHTandsampleabsorbance.BydatamanipulationHTconversionintoabsorbanceandbufferbaselinesubtractionispossible.AlternativelysinglebeamabsorbancescalecanbeusedalreadyinCH2duringdatacollection,loosinghoweverabitthealertingfunctionsofthischannel.Bandwidth(SBW)selectionSettingofslitsshouldbeaslargeaspossible(todecreasenoiselevel),butcompatibletothenaturalbandwidth(NBW)ofthebandstobescanned.AsaruleSBWshouldbekeptatleast1/10oftheNBW,otherwisethebandwillbedistorted.IfNBWisnotknownaseriesoffastsurveyspectraatdifferentSBWwillhelpproperselection.Tradeinofaccuracyversussensitivity(i.e.theuseoflargerthantheoreticalSBW)isoccasionallyrequired.2nminthefarUVregion1nminthearomaticregion(wherefinestructuresmaybepresent),optimalband-pass(aslargeaspossible,but