第二章 園二色性譜 Circular Dichroism (CD)

/ourse/biophysics/cover.htmCircularDichroism

CD圓二色光譜1.什么是圓二色譜?旋光色散譜2.圓二色譜在生物,醫(yī)學研究中的應(yīng)用產(chǎn)生,特征,

structureofmembraneproteincm-1Secondarystructure1624±4sheet1631±3sheet1637±3sheet1645±4Randomcoil1653±4helix1663±4turn1671±3turn1675±5sheet1683±2turn1689±2turn1694±2turnCrystalStructureofMitochondrialRespiratoryMembraneProteinComplexII.Cell,2005,121這是二十五年以來，我國科學家完全立足于“本土”，第一次在該雜志上發(fā)表完整的、系統(tǒng)性的、原創(chuàng)性研究成果hybridorbital

COOHCH3HNH2COOHCH3NH2HCOOHNH2CH3HChiral

LalanineCOOHCH3NH2HDalanineXY線偏振光XYXY頻率相同,方向相反振幅相同,相位相同線偏振光參照面頻率相同,方向相反振幅相同、相位不同線偏振光21參照面yy’axx’RL=tg-1[(|El–Er

|)/(El+Er

)]橢圓偏振光頻率相同,方向相反振幅不同、相位不同參照面合成光特性合成效果比較頻率振幅相位

是否旋轉(zhuǎn)是否橢圓相同相同相同否否相同不同是否不同不同是是左、右旋圓偏振光的合成橢圓偏振光OpticallyActiveSampleChiralRightandLefthandcircularlypolarizedlightPreferentialabsorptionoflefthandpolarized?振動方程Ex=E0x

cos

(ωt+φ0)Ex=E0x

cos

[ω(t-L/v)+φ0]波動方程Ex=Eox

cos

[ωt-(2L/λ)n+φ0]

OrCD(circulardichroism)與振幅

A=lgI0/I=(1/2,303)lnI0/I=ελCLBeer-LanmbertLaw=tg-1[(|El–Er

|)/(El+Er

)]λ

=2.303

Aλ/4(弧度)ΔAλ=Al-

Ar=Δελ

CLLOpticalactiveobjectEx=E0x

cos

[ωt-(2L/λ)n+φ0]

?C:

摩爾濃度

L:

光程(dm)[]l:

[deg?mol-1?cm-1]

or

[deg?cm2?dmol-1]

摩爾橢圓率[]l

(themolarellipticity)[]lMRw

=MRw

[y]l

/100[y]l

(thespecificellipticity)平均殘基橢圓率[]lMRw

(themeanresidue

ellipticity)[]l=Mw

[y]l

/100[

]l

=λ

/CL旋光色散與位相旋光色散ORD(opticalrotatorydispersion)?

nl

nrChiralOpticalactiveobject

=(

/λ)nDn=nl

-nr

a=f(l),弧度ΔΦ=(2

/λ)ΔnLa=[]λLC圓雙折射(Circularbirefringence):

Ex=E0x

cos

[ωt-(2L/λ)n+φ0]

平均殘基旋光度

[m]l(themeanresidue

rotation)[m]l

=[a]l[MRw/100]C:g/mLL

:dmMw:molecularweightMRw:themeanresidueMw[]l

(thespecificrotation)摩爾比旋光度

[j]l

(themolarrotation)[j]l=[a]l[Mw/100][y]l=l/Cw

LPlane(linearly)polarizedlight基本原理(

principle

)RightandLefthandcircularlypolarizedlightOpticallyActiveSamplePreferentialabsorptionoflefthandpolarizedChiralEx=E0x

cos

[ωt-(2l/λ)n+φ0]

Lightnl,nryy’axx’RL[]l=f(l)[a]l=f(l)ORDCDKronig-KramerstransitionrelationshipbetweenCDandORD

andCottoneffect

Cottoneffectl0el0l0+-+-PositivecottoneffectNegativecottoneffectλλλInstrumenthttp://www-/CDofaminoacidandpeptidesAminoacid吲哚基，酚羥基，苯基，二硫鍵，咪唑基，羧基。酪氨酸，色氨酸，苯丙氨酸，胱氨酸，脯氨酸在近紫外區(qū)（240-300nm)對CD譜有貢獻羧基在200-210nm顯示正峰。在波長大于300nm的區(qū)域，包括可見區(qū)域，對CD譜的貢獻主要來自像血紅素一類含有金屬離子的生色團，這一波段CD譜常用于研究金屬離子的氧化態(tài)、配位體以及鏈-鏈相互作用。含有金屬離子的生色團Secondarystructureofmacromoleculea-helixa-helix:190nm(+)208nm,222nm(-)[q]xE-30200210220230240nmb-sheet:195nm(+)

antiparallel:redshiftparallel:blueshift

215-217nm(-)b-sheetUnorderedstructure:(-)below200nm(+)218nmweakbandunorderedb-turn:180-190nm(-)200-205nm(+)225nm(+)band,weakredshiftFarUV（190-240nm)Contentofsecondarystructure

[θ]λ,α、[θ]λ,β、[θ]λ,γ分別為α螺旋、β折疊和非周期結(jié)構(gòu)在波長λ處的橢圓率值fα、fβ、

f

γ分別為α螺旋、β折疊和非周期結(jié)構(gòu)在蛋白質(zhì)中所占重量百分比。圖：三種LRR（lysine-richrepeat)家族肽的CD譜

利用CD譜研究蛋白質(zhì)的構(gòu)象變化TFEUnfolding/folding216nm222nmConformationalvaries190200210220230240250260-1.5-1.0-0.50.00.51.01.52.0

a5in5%SDS/PBSa5in50%TFE/PBSa5inpH7a5in50%TFE/H2Oa5inH2O[|è]X1E-4deg.cm2/dmol-1wavelength(nm)216nm圖

A,Bob1（1-65）和Oct/DNA復(fù)合物的CD譜蛋白質(zhì)-蛋白質(zhì)、蛋白質(zhì)-核酸結(jié)合的研究克山病區(qū)糧飼養(yǎng)的豚鼠心肌線粒體膜的研究模型動物心肌線粒體膜的CD譜（a：病區(qū)糧喂養(yǎng)組；b：病區(qū)糧補加蔬菜喂養(yǎng)組；c：正常對照組）模型動物心肌線粒體細胞色素C氧化酶的CD譜（a：病區(qū)糧喂養(yǎng)組，15周前死亡；b：病區(qū)糧喂養(yǎng)組，存活的；c：正常對照組）煙草花葉病毒侵染膜蛋白磷脂膜誘導(dǎo)的蜂毒素二級結(jié)構(gòu)的變化蜂毒素的CD譜1：蜂毒素插入單層膜的CD譜；2：蜂毒素吸附在單層膜上時的CD譜；3：在Tris-HCL緩沖液中的蜂毒素CD譜1α-螺旋84%，β-折疊1%

無規(guī)卷曲15%2

α-螺旋27%，β-折疊20%

無規(guī)卷曲53%NearUVspectra:toinvestigatethetertiarystructureProteinConcentration:0.5mg/mlCellPathLength:0.5mmStabilizers(Metalions,etc.):

minimumBufferConcentration:

5mMoraslowaspossible

whilemaintainingproteinstabilityTypicalInitialConcentrations:Onemayfindthattheproteinconcentrationneedstobeadjustedtoproducethebestdata.Changingthishasaprofoundeffectonthedata,sosmallincrementsordecrementsarecalledfor.Ifthatdoesnotproducereasonablygooddata,achangeinbuffercompositionmaybenecessary.ItwouldalsobeagoodideatocheckthesampleforunforeseenaggregationviaDynamicLightScattering(DNArepairenzymesareanespeciallygoodexampleofthisbehavior).Ifbufferposesaproblem,cellswithshorterpath(0.1mm)andacorrespondinglyincreasedproteinconcentrationandlongerscantimecanhelp.WhenCDisappliedtoproteinswhatisthemainchromophore(absorbinggroup)thatislookedat?Whatwavelengthrangedoesitabsorbin?DoestheCDspectrumofaproteingivemostinformationaboutthe(i)primarystructure,(ii)secondarystructureor(iii)tertiarystructure?DrawtheCDspectrumofanα-helix.Remembertolabeltheaxesandmarkimportantpointswiththeirvalues.WhyisnitrogengasusedinCDinstruments?DoesCDgiveinformationaboutwherethehelicesareinaprotein?MembraneproteinsaredifficulttostudybyCD.Trueorfalse?Why?思考問題參考文獻《生物物理學》趙南明，周海夢，高教出版社－施普林格出版社2000,ｐ330《園二色性和旋光色散在分子生物學中的應(yīng)用》魯子賢，崔濤，施慶洛，科學出版社1987CircularDichroismandLinearDichroism,AlisonRodgerandBengt

Nordén,OxfordUniversityPress1997《分析儀器手冊》朱良漪，孫亦梁