圆二色光谱CD

圆二色光谱CircularDichroism(CD)洪远凯什么是圆二色谱?旋光色散谱2.圆二色谱在生物,医学研究中的应用产生,特征,WhatisaLight?1888年,Hertz,光波-电磁波16世纪,Newton,光谱17世纪,Huggens,偏振光1881年,Biot,石英能使偏振光的偏振面旋转,电气石圆二色性UVmChiralUVmPlane(linearly)polarizedlightChiralmOpticallyActiveSamplemXYXYEx=E0cosωtEy=E0cos(ωt-/2)Ex2+Ey2=E02Ex=E0cosωtEy=E0cos(ωt+/2)XY频率相同,方向相反振幅相同,相位相同参照面频率相同,方向相反振幅相同、相位不同2参照面1yy’xx’RL频率相同,方向相反振幅不同、相位不同参照面2yy’xx’RL=tg-1[(|El–Er|)/(El+Er)]参照面=(2-1)/2合成光特性合成效果比较频率振幅相位是否旋转是否椭圆相同相同相同否否相同不同是否不同不同是是左、右旋圆偏振光的合成OpticallyActiveSampleChiralPlane(linearly)polarizedlightRightandLefthandcircularlypolarizedlightPreferentialabsorptionoflefthandpolarized?振动方程E=E0cos(ωt+φ0)Ex=E0cos[ω(t-L/v)+φ0]波动方程Ex=Eocos[ωt-(2L/λ)n+φ0]OrωL/v=2Ln/c0=2/λLn圆二色性与振幅A=lgI0/I=(1/2,303)lnI0/I=εCLBeer-LanmbertLaw=2.303A/4(弧度)ΔA=AL-AR=ΔεCLLOpticalactiveobject?C:摩尔浓度L:光程(dm)[]:[deg•mol-1•cm-1]or[deg•cm2•dmol-1]摩尔椭圆率[],[]themolarellipticity[]MRwMRw[y]/100比椭圆率[y]thespecificellipticity平均残基椭圆率[]MRwthemeanresidueellipticity[]Mw[y]/100[y]=/CL旋光色散与位相?Opticalactiveobjectnlnr=(L/λ)n(弧度)n=nl-nrΔΦ=(2L/λ)ΔnLORD(opticalrotatorydispersion)平均残基旋光度[m]themeanresiduerotation[m]=[][MRw/100]C:g/mLL:dmMw:molecularweightMRw:themeanresidueMw比旋光度[y]thespecificrotation摩尔比旋光度[j]themolarrotation[j][][Mw/100][y]/CwLPlane(linearly)polarizedlight基本原理(principle)RightandLefthandcircularlypolarizedlightOpticallyActiveSamplePreferentialabsorptionoflefthandpolarizedChiralLightnl,nryy’xx’RL[]f()[]f()ORDCDKronig-KramerstransitionrelationshipbetweenCDandORDCottoneffectCottoneffect000+-+-PositivecottoneffectNegativecottoneffectλλλInstrument-helix-helix:19xnm(+)208nm,222nm(-)[]xE-30200210220230240nmb-sheet:195nm(+)antiparallel:redshiftparallel:blueshift215-7nm(-)b-sheetUnorderedstructure:(-)below200nm(+)218nmweakbandunorderedb-turn:180-190nm(-)200-205nm(+)225nm(+)band,weakredshift科学研究与开发•中科院某研究所根据HIV-1gp41上的两段七肽重复（heptadrepeat，HR）序列1和2（HR1和HR2）对HIV-1的侵入有抑制作用，进行了HR1和HR2的多螺旋蛋白结构与抗病毒活性的研究，所设计的五螺旋蛋白的基因为HR12121。通过氨基酸连接子（Linker）将三个HR1的基因和二个HR2的基因连接在一起形成一种五螺旋蛋白HR12121的基因。圆二色谱分析证明HR12121和HR212蛋白发现这两种蛋白富含α螺旋结构，这表明HR12121和HR212蛋白非常稳定，不像多肽那样容易降解，并对HIV-1侵染细胞有很高的抑制活性。•目前我们已申请专利“一种抑制囊膜病毒感染的多螺旋蛋白及其编码基因与应用”。囊膜病毒含有许多引起人类重大疾病的病毒，其中抗艾滋病毒的蛋白药物是最重要的。•合作方式：有愿投资者，投资方可获得专利的独家使用权，约需1000-2000万元投资。签订意向书后，具体事宜面议。克山病探究-硒缺乏模型动物心肌线粒体膜的CD谱[]正常对照病区粮病区粮,补加蔬菜烟草花叶病毒侵染膜蛋白引自:吉尚戎等生物物理学报,2004,20Unfolding/folding10203040506070-1.3-1.2-1.1-1.0-0.9[THETA]x1E4temperature(0C)20304050607080-5.0-4.5-4.0-3.5-3.0[THETA]x1E3temperature(0C)216nm222nmConformationalvaries190200210220230240250260-1.5-1.0-0.50.00.51.01.52.0a5in5%SDS/PBSa5in50%TFE/PBSa5inpH7a5in50%TFE/H2Oa5inH2O[¦È]X1E-4deg.cm2/dmol-1wavelength(nm)-0.50.00.51.01.52.02.53.03.54.04.5-3.5-3.0-2.5-2.0-1.5-1.0-0.50.0[THETA]x1E3Gdn.HClconcentration(M)216nmCDspectraofGCN4leucinezipperinthepresenceofdifferentconcentrationsofSDSLeucinezipperpeptidewastreatedwithSDSfor5min.ItsCDspectrawasthenmeasuredatafinalpeptideconcentrationof20μM.ThefinalSDSconcentrationswere0,0.1,0.2,0.3,0.4,0.6,1.0,1.2mMforcurves1,2,3,4,5,6,7,and8,respectively.Curve9isforthecompletelyunfoldedpeptidetreatedwith4Mguanidiniumchloride.Theexperimentswerecarriedoutat20oC.Conformationaltransition:fromalpha-helixrichtobeta-sheetrichPrPcPrPscNearUVspectra:toinvestigatethetertiarystructure250260270280290300310320330340350-250-200-150-100-50050100150BTHETA(degcm2dmol-1)wavelength(nm)振动圆二色谱X射线磁性圆二色吸收谱利用同步辐射的圆偏振和波长连续可调特性，测量铁磁性材料对方向相反的圆偏振光的吸收。由于铁磁性材料对圆偏振光的吸收跃迁不仅取决于轨道量子数，而且与磁量子数有关，因此材料对圆偏振方向相反的光的吸收存在差异，圆二色吸收谱即是研究这种差异所表达出的信息。1.ProteinConcentration:0.5mg/ml2.CellPathLength:0.5mm3.Stabilizers(Metalions,etc.):minimum4.BufferConcentration:5mMoraslowaspossiblewhilemaintainingproteinstabilityTypicalInitialConcentrations:Onemayfindthattheproteinconcentrationneedstobeadjustedtoproducethebestdata.Changingthishasaprofoundeffectonthedata,sosmallincrementsordecrementsarecalledfor.Ifthatdoesnotproducereasonablygooddata,achangeinbuffercompositionmaybenecessary.ItwouldalsobeagoodideatocheckthesampleforunforeseenaggregationviaDynamicLightScattering(DNArepairenzymesareanespeciallygoodexampleofthisbehavior).Ifabsorptionposesaproblem,cellswithshorterpath(0.1mm)andacorrespondinglyincreasedproteinconcentrationandlongerscantimecanhelp.参考文献1.《生物物理学》赵南明，周海梦，高教出版社－施普林格出版社2000,ｐ3302.《园二色性和旋光色散在分子生物学中的应用》鲁子贤，崔涛，施庆洛，科学出版社19873.CircularDichroismandLinearDichroism,AlisonRodgerandBengtNordén,OxfordUniversityPress19974.《分析仪器手册》朱良漪，孙亦梁，陈耕燕，化学工业出版社1997,p2475.CircularDichroismandOpticalRotaryDispersionofProteinsandPolypeptides,A.J.Alder,N.J.GreenfieldandG.D.Fasman,Meth.Enzymology,27,675(1973).6.ComputedCircularDichroismSpectrafortheEvaluationofProteinConformation,N.GreenfieldandG.D.Fasman,Biochemistry,8(10),4108(1996)7.C.R.CantorandP.R.Schimmel,BiophysicalChemistry,Vol.2,Chapter8(1980)8.EffectofTrifluoroethanolonProteinSecondaryStructure,F.D.Soennichsen,J.E.VanEyk,R.S.HodgesandB.D.Sykes