圆二色光谱CD

1、What is a Light?What is a Light? 18881888年年,Hertz,Hertz,光波光波- -电磁波电磁波日月之日月之辉辉16世世纪纪,Newton,光光谱谱17世世纪纪,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转转, 电电气石气石圆圆二色性二色性UV m Chiral UV m Plane(linearly) polarized lightChiral 椭圆偏振光椭圆偏振光 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频率相同,方向相反振幅相同,相位相同参照面频率相同,方向相反振幅相同、相位不同2参照面1 yyxxRL椭圆偏振光椭圆偏振光频率相同,方向相反振幅不同、相位不同参照面21 yyxxRL = tg-1(|El Er |)/(El + Er )参照面12=(2-1)/2 合成光特性合成光特性 合成效果合成效果比比较较频频率率 振幅 相位 是否旋旋转转是否椭圆椭圆相同相同 相同 否 否相同 不同 是 否不同 不同 是 是左、右左、右旋旋圆圆偏振光偏振光的的合成合成椭圆偏振光椭圆偏振

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 ?振振动动方程方程= =E E0 0 coscos ( ( t+t+ 0 0 ) )= =E E0 0

4、coscos ( (t- t-L/vL/v)+)+ 0 0 波波动动方程方程= =E Eo o coscos t t-(2-(2 L/)n+ + 0 0 L/v=2L n/c0= 2 / Ln圆圆二色性二色性与振幅与振幅 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、摩尔浓度摩尔浓度 L : 光程光程 ( (d dm)m) : : deg mol-1 cm-1 or deg cm2 dmol-1 摩尔椭圆率摩尔椭圆率 , the molar ellipticity MRw MRw y y /100比椭圆率比椭圆率y y the specific ellipticity平均残基椭圆率平均残基椭圆率 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)平均残基旋光度平均残基旋光度 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摩尔比旋光度摩尔比旋光度 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科学研究与开发科学研究与开发 中科院某研究所根据HIV-1gp41上的两段七肽重复（heptad repeat，HR）序列1和2（HR1和HR2）对HIV-1的侵入有抑制作用，进行了HR1和HR2的多螺旋蛋白结构与抗病毒活性的研究，所设计的五螺旋蛋白的基因为HR12121。通过氨基酸连接子（Linker）将三个HR1的基因和二个HR2的基因连接在一起形成一种五螺旋蛋白HR12121的基因。圆二色谱分析证明HR12121和HR212蛋白发现这两种蛋白富含螺旋结构，这表明HR12121和HR212蛋白非

11、常稳定，不像多肽那样容易降解，并对HIV-1侵染细胞有很高的抑制活性。 目前我们已申请专利“一种抑制囊膜病毒感染的多螺旋蛋白及其编码基因与应用”。囊膜病毒含有许多引起人类重大疾病的病毒，其中抗艾滋病毒的蛋白药物是最重要的。 合作方式：有愿投资者，投资方可获得专利的独家使用权，约需1000-2000万元投资。签订意向书后，具体事宜面议。 克山病探究-硒缺乏模型动物心肌线粒体膜的CD谱正常对照正常对照病区粮病区粮病区粮病区粮,补加蔬菜补加蔬菜引自引自:吉尚戎等吉尚戎等生物物理学生物物理学报报,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射射线线磁性磁性圆圆二色吸收二色吸收谱谱利用同步辐射的圆偏振和波长连续可调特性，测量铁磁性材料对方向相反的圆偏振光的吸收。由于铁磁性材料对圆偏振光的吸收跃迁不仅取决于轨道量子数，而且与磁量子数有关，因此材料对圆偏振方向

16、相反的光的吸收存在差异，圆二色吸收谱即是研究这种差异所表达出的信息。 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.赵南明，周海梦，高教出版社施普林格出版社赵南明，周海梦，高

19、教出版社施普林格出版社2000,2000,3303302.2.鲁子贤，崔涛，施庆洛，科学鲁子贤，崔涛，施庆洛，科学出版社出版社19871987, , Alison Rodger and Bengt Alison Rodger and Bengt NordnNordn, Oxford University Press 1997, Oxford University Press 19974.4.朱良漪，孙亦梁，陈耕燕，化学工业出版社朱良漪，孙亦梁，陈耕燕，化学工业出版社 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页倒数13，14行漏印了一段，应改为： 偶极子置于离子所偶极子置于离子所产产生的静生的静电场电场中中时时，二者，二者间间的的相互作用称相互作用称为电为电荷荷-偶极相互作用。例如：偶极相互作用。例如：K+离子和其外离子和其外围围水化水化层层中的水分子中的水分子（是一种偶极子）的作用。（是一种偶极子）的作用。这类这类相互作用能和距离的平相互作用能和距离的平方成反比。方成反比。 偶极子置于另一偶极子所偶极子置于另一偶极子所产产生的静生的静电场电场中中时时，二者，二者间间的相互作用的相互作用称称为为偶极偶极-偶极相互

25、作用，其相互作用能和距离的三偶极相互作用，其相互作用能和距离的三次方成反比。次方成反比。P53页第8行“磷脂分子的形状磷脂分子的形状”，应改为“脂的多型性脂的多型性” ”第20行 “biomolecular”，应改为“bilayer”P75页倒数第12行公式（5-10）中“Km”，应改为“km”P77页第8行中“离子梯度”，应改为“离子浓度梯度” ”P95页图注第3行中“色侧链”，应改为“深灰色侧链” ”P96页图（6-9）注第13行中“浅蓝线”，应改为“深蓝线” ”第15行中“深蓝线”，应改为“浅蓝线” ” 医学生物物理学医学生物物理学勘误勘误P113标题标题 “Adhension”，应改为

26、“Adhesion”P114第13行 “viscsiometer”，应改为“viscosimeter”P132页文献5“acetglchine”，应改为“acetylcholine”“elecfron”，应改为“electron”P189页倒数第16行“另别一重”，应改为“另外一重” ”P201页倒数第2行“2p”，应改为“2”P202页倒数第1行中二个“长度”，应改为“振幅” ”P272页标题 “主要其中”，应改为“主要英中” ”右右边边“ “biomolecular”，应改为“bilayer”P273页右边倒6行“excited”，应改为“excitation”倒5行“excitation”，应改为“excited”P276页左边第3行“enhanced”，应改为“enhancement”右边第8行“photoxdation”，应改为“photooxidation”P278页左边倒数第7行“trplet”，应改为“triplet”倒数第6行“扭曲”，应改为“扭曲振动” ” 1.What is meant by “far UV” light?2.When CD is applied to proteins what is the main chromophore (absorbing group) that