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(zhuǎn)轉(zhuǎn), 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 coscos t t=E0 cos (t-

2、 /2)E Ey y2 2 =E=E0 02 2= =E E0 0 coscos 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(zhuǎn)轉(zhuǎn)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偏振

3、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 coscos ( ( t+t+ 0 0 ) )= =E E0 0

4、coscos ( (t- t-L/vL/v)+)+ 0 0 波波動動方程方程= =E Eo o coscos 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 coscos t- t-( (2 L /)n + + 0 0 ?C:

5、摩爾濃度摩爾濃度 L : 光程光程 ( (d dm)m) : : 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 coscos 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 : molecular weight MMRwRw: the mean residue M: the mean residue Mw w比旋光度比旋光度 y y th

7、e 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 absorption

8、absorption of left of left hand hand polarized polarized Chiral= =E E0 x0 x coscos 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-structu

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: 180-190nm

10、(-) 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(jié)構(gòu)與抗病毒活性的研究，所設(shè)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(jié)構(gòu)，這表明HR12121和HR212蛋白非

11、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(yīng)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(quán)，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/folding10203040506070-1.3-1.2-1.1-1.

12、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)-0.50.00.51.01.52.0

13、2.53.03.54.04.5-3.5-3.0-2.5-2.0-1.5-1.0-0.50.0THETAx1E3Gdn.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 M. T

14、he 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-helix ri

15、ch 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(diào)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(guān)，因此材料對圓偏振方向

16、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。 1. Protein Concentration: 0.5 mg/ml2. Cell Path Length: 0.5 mm3. Stabilizers (Metal ions, etc.): minimum4. Buffer Concentration : 5 mM or as low as possible while maintaining protein stabilityTypical Initial Concentrations:One may find that the protein concentratio

17、n needs 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 unf

18、oreseen 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.1.1.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

19、教出版社施普林格出版社2000,2000,3303302.2.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 NordnNordn, Oxford University Press 1997, Oxford University Press 19974.4.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, 1997, p 247p 2475.5.Circular Circular DichroismDichroism a

20、nd Optical Rotary Dispersion of Proteins and and Optical Rotary Dispersion of Proteins and Polypeptides, A.J.Alder, N.J.Greenfield and Polypeptides, A.J.Alder, N.J.Greenfield and G.D.FasmanG.D.Fasman, , MethMeth. . EnzymologyEnzymology ,27, 675 (1973). ,27, 675 (1973). 6.6.Computed Circular Computed

21、 Circular DichroismDichroism Spectra for the Evaluation of Protein Spectra for the Evaluation of Protein Conformation, N.Greenfield and Conformation, N.Greenfield and G.D.FasmanG.D.Fasman, , Biochemistry,Biochemistry, 8 (10), 4108 8 (10), 4108 (1996) (1996) 7.7.C.R.Cantor and C.R.Cantor and P.R.Schi

22、mmelP.R.Schimmel, , Biophysical ChemistryBiophysical Chemistry, Vol.2, Chapter 8 , Vol.2, Chapter 8 (1980) (1980) 8.8.Effect of Effect of TrifluoroethanolTrifluoroethanol on Protein Secondary Structure, on Protein Secondary Structure, F.D.SoennichsenF.D.Soennichsen, , J.E.VanEykJ.E.VanEyk, R.S.Hodge

23、s and B.D.Sykes, , R.S.Hodges and B.D.Sykes, Biochemistry,Biochemistry, 31 (37),8791 (1992) 31 (37),8791 (1992) 9.9.Protein Secondary Structure and Circular Protein Secondary Structure and Circular DichroismDichroism : A Practical Guide, : A Practical Guide, W.C.JohnsonW.C.Johnson, PROTEINS, , PROTE

24、INS, 7, 205-214 (1990) 7, 205-214 (1990)P30頁倒數(shù)13，14行漏印了一段，應(yīng)改為： 偶極子置于離子所偶極子置于離子所產(chǎn)產(chǎn)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(chǎn)產(chǎn)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

25、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(yīng)改為“脂的多型性脂的多型性” ”第20行 “biomolecular”，應(yīng)改為“bilayer”P75頁倒數(shù)第12行公式（5-10）中“Km”，應(yīng)改為“km”P77頁第8行中“離子梯度”，應(yīng)改為“離子濃度梯度” ”P95頁圖注第3行中“色側(cè)鏈”，應(yīng)改為“深灰色側(cè)鏈” ”P96頁圖（6-9）注第13行中“淺藍線”，應(yīng)改為“深藍線” ”第15行中“深藍線”，應(yīng)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(yīng)改為

26、“Adhesion”P114第13行 “viscsiometer”，應(yīng)改為“viscosimeter”P132頁文獻5“acetglchine”，應(yīng)改為“acetylcholine”“elecfron”，應(yīng)改為“electron”P189頁倒數(shù)第16行“另別一重”，應(yīng)改為“另外一重” ”P201頁倒數(shù)第2行“2p”，應(yīng)改為“2”P202頁倒數(shù)第1行中二個“長度”，應(yīng)改為“振幅” ”P272頁標題 “主要其中”，應(yīng)改為“主要英中” ”右右邊邊“ “biomolecular”，應(yīng)改為“bilayer”P273頁右邊倒6行“excited”，應(yīng)改為“excitation”倒5行“excitation”，應(yīng)改為“excited”P276頁左邊第3行“enhanced”，應(yīng)改為“enhancement”右邊第8行“photoxdation”，應(yīng)改為“photooxidation”P278頁左邊倒數(shù)第7行“trplet”，應(yīng)改為“triplet”倒數(shù)第6行“扭曲”，應(yīng)改為“扭曲振動” ” 1.What is meant by “far UV” light?2.When CD is applied to proteins what is the main chromophore (absorbing group) that