第二章園二色性譜circulardichroism(cd)

1、,,snl.bjmu.edu.cn/ourse/biophysics/cover.htm,Circular Dichroism 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(xué) 研究中的應(yīng)用,產(chǎn)生,,特征,,structure of membrane protein,Crystal Structure of Mitochondrial Respiratory Membrane Protein

2、Complex II. Cell, 2005,121,這是二十五年以來，我國科學(xué)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,hybrid orbital,,Chiral,L alanine,D alanine,,,,,,,X,Y,,,,,,,線偏振光,,,,,,,,X,Y,,,,,,,,,,X,Y,,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

3、相反振幅相同、相位不同,,?,,,,,,,線偏振光,,,?2,?1,參照面,,,,,,,,,,,,,,,,,?,y,y’,a,x,x’,R,L,? = 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,,左、右旋圓偏振光的合成,橢圓偏振光,,,,,Optically Active Sample,Chiral,,,Right and L

4、eft hand circularly polarized light,,,Preferential absorption of left hand polarized,?,?,,,,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-(2?L/λ)n+φ0],Or,,,,CD (circular dichroism)與振幅,A= lgI0/

5、I=(1/2,303)lnI0/I=ελCL,Beer-Lanmbert Law,,,? = tg-1[(|El – Er |)/(El + Er )],?λ =2.303 ? Aλ/4 (弧度),ΔAλ=Al - Ar =Δελ C L,,,,,L,,,Optical active object,Ex= E0x cos [ωt-(2?L /λ)n+φ0],,,,?,,C: 摩爾濃度 L : 光程 (dm)[? ]l:

6、 [deg ? mol-1 ? cm-1] or [deg ? cm2 ? dmol-1],摩爾橢圓率[? ]l (the molar ellipticity),[? ]lMRw = MRw [y]l /100,[y]l (the specific ellipticity),平均殘基橢圓率[? ]lMRw (the mean residue ellipticity),[? ]l = Mw [

7、y]l /100,,[? ]l = ?λ /CL,旋光色散與位相,旋光色散ORD (optical rotatory dispersion),,,,,?,nl nr,Chiral,Optical active object,? = (? /λ) ?n,Dn = nl - nr,a = f (l),弧度,ΔΦ=(2? /λ)Δn,,,,,L,a =[?]λLC,,圓雙折射(Circular birefringence):,,Ex

8、=E0x cos [ωt-(2?L/λ)n+φ0],,平均殘基旋光度 [m]l (the mean residue rotation),[m]l = [a]l ?[MRw/100],C : g/mLL : dmMw: molecular weightMRw: the mean residue Mw,[?]l (the specific rotatio

9、n),摩爾比旋光度 [j]l (the molar rotation),[j]l = [a]l ?[Mw/100],[y]l = ?l/ Cw L,,,,,Plane(linearly) polarized light,基本原理( principle ),Right and Left hand circularly polarized light,Optically Active Sample,Preferential

10、 absorption of left hand polarized,Chiral,,,,Ex=E0x cos [ωt-(2?l /λ)n+φ0],,,,,,Light,nl , nr,,,,,,,,,,,,,,,,,?,y,y’,,a,x,x’,,R,,L,[? ]l = f (l),[a]l = f (l),,ORD,CD,,Kronig-Kramerstransition,relationship between CD and

11、ORD and Cotton effect,Cotton effect,,,,,l0,e,,,,,l0,,,,,,l0,,+,-,+,-,Positive cottoneffect,Negative cottoneffect,λ,λ,λ,Instrument,http://www-structure.llnl.gov/,CD of amino acid and peptides,Amino acid,吲哚基，

12、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,Secondary structure of macromolecule,a-hel

13、ix,a-helix: 190 nm (+) 208nm, 222nm (-),b-sheet: 195nm (+) antiparallel: red shift parallel: blue shift 215-217nm (-),,b-sheet,Unordered structure: (-) below 200nm (+) 218nm weak band,,unordered,b-turn: 180-1

14、90nm (-) 200-205nm (+) 225nm (+) band, weak red shift,Far UV（190-240nm),Content of secondary structure,,,,[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-rich

15、 repeat)家族肽的CD譜,利用CD譜研究蛋白質(zhì)的構(gòu)象變化,,TFE,Unfolding/folding,216nm,222nm,Conformational varies,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

16、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%,Near UV spectra

17、: to investigate the tertiary structure,Protein Concentration: 0.5 mg/mlCell Path Length: 0.5 mmStabilizers (Metal ions, etc.): minimumBuffer Concentration : 5 mM or as low as possible while maintaining protein stabi

18、lity,Typical Initial Concentrations:,One may find that the protein concentration needs to be adjusted to produce the best data. Changing this has a profound effect on the data, so small increments or decrements are calle

19、d for. If that does not produce reasonably good data, a change in buffer composition may be necessary. It would also be a good idea to check the sample for unforeseen aggregation via Dynamic Light Scattering (DNA repair

20、enzymes are an especially good example of this behavior). If buffer poses a problem, cells with shorter path (0.1 mm) and a correspondingly increased protein concentration and longer scan time can help.,When CD is applie

21、d to proteins what is the main chromophore (absorbing group) that is looked at? What wavelength range does it absorb in?Does the CD spectrum of a protein give most information about the (i) primary structure, (ii) secon

22、dary structure or (iii) tertiary structure?Draw the CD spectrum of an α-helix. Remember to label the axes and mark important points with their values.Why is nitrogen gas used in CD instruments?Does CD give information

23、 about where the helices are in a protein?Membrane proteins are difficult to study by CD. True or false? Why?,思 考 問 題,參考文獻,《生物物理學(xué)》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(xué)中的應(yīng)用》魯子賢，崔濤，施慶洛，科學(xué)出版社　1987Circular Dichr

24、oism and Linear Dichroism, Alison Rodger and Bengt Nordén, Oxford University Press 1997《分析儀器手冊》朱良漪，孫亦梁，陳耕燕，化學(xué)工業(yè)出版社 1997, p 247Circular Dichroism and Optical Rotary Dispersion of Proteins and Polypeptides, A.J.Alde

25、r, N.J.Greenfield and G.D.Fasman, Meth. Enzymology ,27, 675 (1973). Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation, N.Greenfield and G.D.Fasman, Biochemistry, 8 (10), 4108 (1996) C.R.Can

26、tor and P.R.Schimmel, Biophysical Chemistry, Vol.2, Chapter 8 (1980) Effect of Trifluoroethanol on Protein Secondary Structure, F.D.Soennichsen, J.E.VanEyk, R.S.Hodges and B.D.Sykes, Biochemistry, 31 (37),8791 (1992) P