圓二色光譜CD

1、What is a Light?What is a Light? 18881888年年,Hertz,Hertz,光波光波- -電磁波電磁波日月之日月之輝輝16世世紀紀,Newton,光光譜譜17世世紀紀,Huggens,偏振光偏振光1881年年,Biot,石英能使偏振光的石英能使偏振光的偏振偏振面旋面旋轉轉, 電電氣石氣石圓圓二色性二色性UV m Chiral UV m Plane(linearly) polarized lightChiral 橢圓偏振光橢圓偏振光 m Optically Active Sample m ？XYXY= =E E0 0 cos cos t t=E0 cos (t

2、- /2)E Ey y2 2 =E=E0 02 2= =E E0 0 cos cos t t=E0 cos (t+ /2)XY頻率相同,方向相反振幅相同,相位相同參照面頻率相同,方向相反振幅相同、相位不同2參照面1 yyxxRL橢圓偏振光橢圓偏振光頻率相同,方向相反振幅不同、相位不同參照面21 yyxxRL = tg-1(|El Er |)/(El + Er )參照面12=(2-1)/2 合成光特性合成光特性 合成效果合成效果比比較較頻頻率率 振幅 相位 是否旋旋轉轉是否橢圓橢圓相同相同 相同 否 否相同 不同 是 否不同 不同 是 是左、右左、右旋旋圓圓偏振光偏振光的的合成合成橢圓偏振光橢圓

3、偏振光Optically Active SampleChiral Plane(linearly) polarized lightRight and Right and Left hand Left hand circularly circularly polarized polarized lightlightPreferential Preferential absorption absorption of left of left hand hand polarized polarized ?振振動動方程方程= =E E0 0 cos cos ( ( t+t+ 0 0 ) )= =E E0

4、 0 cos cos (t-(t-L/vL/v)+)+ 0 0 波波動動方程方程= =E Eo o cos cos t t-(2-(2 L/)n+ + 0 0 L/v=2L n/c0= 2 / Ln圓圓二色性二色性與振幅與振幅 A A= lgI= lgI0 0/I /I =(1/2,303)lnI =(1/2,303)lnI0 0/I /I = = C CL LBeer-Lanmbert Law =2.303 A/4 (弧度弧度)A=A=AL-L- AR = C LLOptical active object= = E E0 0 cos cos t- t-( (2 L /)n + + 0 0

5、?C: 摩爾濃度摩爾濃度 L : 光程光程 ( (dm)dm) : : deg mol-1 cm-1 or deg cm2 dmol-1 摩爾橢圓率摩爾橢圓率 , the molar ellipticity MRw MRw y y /100比橢圓率比橢圓率y y the specific ellipticity平均殘基橢圓率平均殘基橢圓率 MRw the mean residue ellipticity Mw y y /100y y = /CL?Optical active object nl nr = ( = ( L /) ) n (n (弧度) ) n = nl - nr =(2=(2 L

6、 / / ) ) n nL= =E E0 0 cos cos t t-( -(2 L/)n+ + 0 0 ORD (optical rotatory dispersion)平均殘基旋光度平均殘基旋光度 mm the mean residue the mean residue rotationrotation mm = MRw/100C C : g/mL : g/mL L L : dm: dmMMw w: molecular weight M: molecular weight MRwRw: the mean residue M: the mean residue Mw w比旋光度比旋光度 y

7、y the specific rotationthe specific rotation摩爾比旋光度摩爾比旋光度 j j the molar rotationthe molar rotation j j Mw/100y y / / Cw L Plane(linearly) polarized lightRight and Right and Left hand Left hand circularly circularly polarized polarized lightlightOptically Active SamplePreferential Preferential absorpt

8、ion absorption of left of left hand hand polarized polarized Chiral= =E E0 x0 x cos cos t t-( -(2 l /)n+ + 0 0 Light nl , nr yyxxRL f () f ()ORDCDKronig-Kramerstransitionrelationship between CD and ORD Cotton effect Cotton effect000+-+-Positive cottoneffectNegative cottoneffectInstrumenthttp:/www-st

9、/CD applicationSecondary structure of macromolecule-helix -helix: 19x nm (+) 208nm, 222nm (-) x E-30200210220 230 240nmb b-sheet: 195nm (+) antiparallel: red shift parallel: blue shift 215-7nm (-) b-sheetUnordered structure: (-) below 200nm (+) 218nm weak bandunorderedb b-turn: 180-1

10、90nm (-) 200-205nm (+) 225nm (+) band, weak red shift科學研究與開發(fā)科學研究與開發(fā) 中科院某研究所根據(jù)HIV-1gp41上的兩段七肽重復（heptad repeat，HR）序列1和2（HR1和HR2）對HIV-1的侵入有抑制作用，進行了HR1和HR2的多螺旋蛋白結構與抗病毒活性的研究，所設計的五螺旋蛋白的基因為HR12121。通過氨基酸連接子（Linker）將三個HR1的基因和二個HR2的基因連接在一起形成一種五螺旋蛋白HR12121的基因。圓二色譜分析證明HR12121和HR212蛋白發(fā)現(xiàn)這兩種蛋白富含螺旋結構，這表明HR12121和HR2

11、12蛋白非常穩(wěn)定，不像多肽那樣容易降解，并對HIV-1侵染細胞有很高的抑制活性。 目前我們已申請專利“一種抑制囊膜病毒感染的多螺旋蛋白及其編碼基因與應用”。囊膜病毒含有許多引起人類重大疾病的病毒，其中抗艾滋病毒的蛋白藥物是最重要的。 合作方式：有愿投資者，投資方可獲得專利的獨家使用權，約需1000-2000萬元投資。簽訂意向書后，具體事宜面議。 克山病探究-硒缺乏模型動物心肌線粒體膜的CD譜正常對照正常對照病區(qū)糧病區(qū)糧病區(qū)糧病區(qū)糧,補加蔬菜補加蔬菜引自引自:吉尚戎等吉尚戎等生物物理學生物物理學報報,2004,20Unfolding/folding10203040506070-1.3-1.2-1

12、.1-1.0-0.9THETAx1E4temperature (0C)20304050607080-5.0-4.5-4.0-3.5-3.0THETAx1E3temperature (0C)216nm222nmConformational varies190200210220230240250260-1.5-1.0-0.50.00.51.01.52.0 a5 in 5 % SDS/PBS a5 in 50 % TFE/PBS a5 in pH7 a5 in 50 % TFE/H2O a5 in H2OX1E-4 deg.cm2/dmol-1wavelength(nm)-0.50.00.51.01

13、.52.02.53.03.54.04.5-3.5-3.0-2.5-2.0-1.5-1.0-0.50.0THETAx1E3Gdn.HCl concentration (M)216nmCD spectra of GCN4 leucine zipper in the presence of different concentrations of SDSLeucine zipper peptide was treated with SDS for 5 min. Its CD spectra was then measured at a final peptide concentration of 20

14、 M. The final SDS concentrations were 0, 0.1, 0.2, 0.3, 0.4, 0.6, 1.0, 1.2 mM for curves 1, 2, 3, 4, 5, 6, 7, and 8, respectively. Curve 9 is for the completely unfolded peptide treated with 4 M guanidinium chloride. The experiments were carried out at 20 oC.Conformational transition: from alpha-hel

15、ix rich to beta-sheet richPrPc PrPscScout Near UV spectra: to investigate the tertiary structure250260270280290300310320330340350-250-200-150-100-50050100150BTHETA (deg cm2 dmol-1)wavelength (nm)X射射線線磁性磁性圓圓二色吸收二色吸收譜譜利用同步輻射的圓偏振和波長連續(xù)可調特性，測量鐵磁性材料對方向相反的圓偏振光的吸收。由于鐵磁性材料對圓偏振光的吸收躍遷不僅取決于軌道量子數(shù)，而且與磁量子數(shù)有關，因此材料對

16、圓偏振方向相反的光的吸收存在差異，圓二色吸收譜即是研究這種差異所表達出的信息。 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 stabilityTypical Initial Concentrations:One may find that the protein concentration needs

17、 to be adjusted to produce the best data. Changing this has a profound effect on the data, so small increments or decrements are called 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

18、 aggregation via Dynamic Light Scattering (DNA repair enzymes are an especially good example of this behavior). If absorption poses a problem, cells with shorter path (0.1 mm) and a correspondingly increased protein concentration and longer scan time can help.趙南明，周海夢，高教出版社施普林格出版社趙南明，周海夢，高教出版社施普林格出版社

19、2000,2000,330330魯子賢，崔濤，施慶洛，科學魯子賢，崔濤，施慶洛，科學出版社出版社19871987, , Alison Rodger and Bengt Alison Rodger and Bengt Nordn, Oxford University Press 1997Nordn, Oxford University Press 1997朱良漪，孫亦梁，陳耕燕，化學工業(yè)出版社朱良漪，孫亦梁，陳耕燕，化學工業(yè)出版社 1997, p 247 1997, p 247Circular Dichroism and Optical Rotary Dispersion of Proteins

20、 and Circular Dichroism and Optical Rotary Dispersion of Proteins and Polypeptides, A.J.Alder, N.J.Greenfield and G.D.Fasman, Polypeptides, A.J.Alder, N.J.Greenfield and G.D.Fasman, Meth. Meth. EnzymologyEnzymology ,27, 675 (1973). ,27, 675 (1973). Computed Circular Dichroism Spectra for the Evaluat

21、ion of Protein Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation, N.Greenfield and G.D.Fasman, Conformation, N.Greenfield and G.D.Fasman, Biochemistry,Biochemistry, 8 (10), 4108 8 (10), 4108 (1996) (1996) C.R.Cantor and P.R.Schimmel, C.R.Cantor and P.R.Schimmel, Biophysi

22、cal ChemistryBiophysical Chemistry, Vol.2, Chapter 8 , Vol.2, Chapter 8 (1980) (1980) Effect of Trifluoroethanol on Protein Secondary Structure, F.D.Soennichsen, Effect of Trifluoroethanol on Protein Secondary Structure, F.D.Soennichsen, J.E.VanEyk, R.S.Hodges and B.D.Sykes, J.E.VanEyk, R.S.Hodges a

23、nd B.D.Sykes, Biochemistry,Biochemistry, 31 (37),8791 (1992) 31 (37),8791 (1992) Protein Secondary Structure and Circular Dichroism : A Practical Guide, Protein Secondary Structure and Circular Dichroism : A Practical Guide, W.C.JohnsonW.C.Johnson, PROTEINS, , PROTEINS, 7, 205-214 (1990) 7, 205-214

24、(1990)P30頁倒數(shù)13，14行漏印了一段，應改為： 偶極子置于離子所偶極子置于離子所產產生的靜生的靜電場電場中中時時，二者，二者間間的的相互作用稱相互作用稱為電為電荷荷-偶極相互作用。例如：偶極相互作用。例如：K+離子和其外離子和其外圍圍水化水化層層中的水分子中的水分子（是一種偶極子）的作用。（是一種偶極子）的作用。這類這類相互作用能和距離的平相互作用能和距離的平方成反比。方成反比。 偶極子置于另一偶極子所偶極子置于另一偶極子所產產生的靜生的靜電場電場中中時時，二者，二者間間的相互作用的相互作用稱稱為為偶極偶極-偶極相互作用，其相互作用能和距離的三偶極相互作用，其相互作用能和距離的三次方

25、成反比。次方成反比。P53頁第8行“磷脂分子的形狀磷脂分子的形狀”，應改為“脂的多型性脂的多型性” ”第20行 “biomolecular”，應改為“bilayer”P75頁倒數(shù)第12行公式（5-10）中“Km”，應改為“km”P77頁第8行中“離子梯度”，應改為“離子濃度梯度” ”P95頁圖注第3行中“色側鏈”，應改為“深灰色側鏈” ”P96頁圖（6-9）注第13行中“淺藍線”，應改為“深藍線” ”第15行中“深藍線”，應改為“淺藍線” ” 醫(yī)學生物物理學醫(yī)學生物物理學勘誤勘誤P113標題標題 “Adhension”，應改為“Adhesion”P114第13行 “viscsiometer”，

26、應改為“viscosimeter”P132頁文獻5“acetglchine”，應改為“acetylcholine”“elecfron”，應改為“electron”P189頁倒數(shù)第16行“另別一重”，應改為“另外一重” ”P201頁倒數(shù)第2行“2p”，應改為“2”P202頁倒數(shù)第1行中二個“長度”，應改為“振幅” ”P272頁標題 “主要其中”，應改為“主要英中” ”右右邊邊“ “biomolecular”，應改為“bilayer”P273頁右邊倒6行“excited”，應改為“excitation”倒5行“excitation”，應改為“excited”P276頁左邊第3行“enhanced”，應改為“enhancement”右邊第8行“photoxdation”，應改為“photooxidation”P278頁左邊倒數(shù)第7行“trplet”，應改為“triplet”倒數(shù)第6行“扭曲”，應改為“扭曲振動” ” What is meant by “far UV” light?When CD is applied to proteins what is the main chromophore (absorbing grou