第二章 园二色性谱 Circular Dichroism (CD)

/ourse/biophysics/cover.htmCircularDichroism

CD圆二色光谱1.什么是圆二色谱?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,

structureofmembraneproteincm-1Secondarystructure1624±4sheet1631±3sheet1637±3sheet1645±4Randomcoil1653±4helix1663±4turn1671±3turn1675±5sheet1683±2turn1689±2turn1694±2turnCrystalStructureofMitochondrialRespiratoryMembraneProteinComplexII.Cell,2005,121这是二十五年以来，我国科学家完全立足于“本土”，第一次在该杂志上发表完整的、系统性的、原创性研究成果hybridorbital

COOHCH3HNH2COOHCH3NH2HCOOHNH2CH3HChiral

LalanineCOOHCH3NH2HDalanineXY线偏振光XYXY频率相同,方向相反振幅相同,相位相同线偏振光参照面频率相同,方向相反振幅相同、相位不同线偏振光21参照面yy’axx’RL=tg-1[(|El–Er

|)/(El+Er

)]椭圆偏振光频率相同,方向相反振幅不同、相位不同参照面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椭圆偏振光OpticallyActiveSampleChiralRightandLefthandcircularlypolarizedlightPreferentialabsorptionoflefthandpolarized?振动方程Ex=E0x

cos

(ωt+φ0)Ex=E0x

cos

[ω(t-L/v)+φ0]波动方程Ex=Eox

cos

[ωt-(2L/λ)n+φ0]

OrCD(circulardichroism)与振幅

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:

摩尔浓度

L:

光程(dm)[]l:

[deg•mol-1•cm-1]

or

[deg•cm2•dmol-1]

摩尔椭圆率[]l

(themolarellipticity)[]lMRw

=MRw

[y]l

/100[y]l

(thespecificellipticity)平均残基椭圆率[]lMRw

(themeanresidue

ellipticity)[]l=Mw

[y]l

/100[

]l

=λ

/CL旋光色散与位相旋光色散ORD(opticalrotatorydispersion)?

nl

nrChiralOpticalactiveobject

=(

/λ)nDn=nl

-nr

a=f(l),弧度ΔΦ=(2

/λ)ΔnLa=[]λLC圆双折射(Circularbirefringence):

Ex=E0x

cos

[ωt-(2L/λ)n+φ0]

平均残基旋光度

[m]l(themeanresidue

rotation)[m]l

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

:dmMw:molecularweightMRw:themeanresidueMw[]l

(thespecificrotation)摩尔比旋光度

[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，酚羟基，苯基，二硫键，咪唑基，羧基。酪氨酸，色氨酸，苯丙氨酸，胱氨酸，脯氨酸在近紫外区（240-300nm)对CD谱有贡献羧基在200-210nm显示正峰。在波长大于300nm的区域，包括可见区域，对CD谱的贡献主要来自像血红素一类含有金属离子的生色团，这一波段CD谱常用于研究金属离子的氧化态、配位体以及链-链相互作用。含有金属离子的生色团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分别为α螺旋、β折叠和非周期结构在波长λ处的椭圆率值fα、fβ、

f

γ分别为α螺旋、β折叠和非周期结构在蛋白质中所占重量百分比。图：三种LRR（lysine-richrepeat)家族肽的CD谱

利用CD谱研究蛋白质的构象变化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图

A,Bob1（1-65）和Oct/DNA复合物的CD谱蛋白质-蛋白质、蛋白质-核酸结合的研究克山病区粮饲养的豚鼠心肌线粒体膜的研究模型动物心肌线粒体膜的CD谱（a：病区粮喂养组；b：病区粮补加蔬菜喂养组；c：正常对照组）模型动物心肌线粒体细胞色素C氧化酶的CD谱（a：病区粮喂养组，15周前死亡；b：病区粮喂养组，存活的；c：正常对照组）烟草花叶病毒侵染膜蛋白磷脂膜诱导的蜂毒素二级结构的变化蜂毒素的CD谱1：蜂毒素插入单层膜的CD谱；2：蜂毒素吸附在单层膜上时的CD谱；3：在Tris-HCL缓冲液中的蜂毒素CD谱1α-螺旋84%，β-折叠1%

无规卷曲15%2

α-螺旋27%，β-折叠20%

无规卷曲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?思考问题参考文献《生物物理学》赵南明，周海梦，高教出版社－施普林格出版社2000,ｐ330《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1987CircularDichroismandLinearDichroism,AlisonRodgerandBengt

Nordén,OxfordUniversityPress1997《分析仪器手册》朱良漪，孙亦梁