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1、圆二色光谱圆二色光谱CDCD1.什么是圆什么是圆二色谱二色谱?旋光色散谱旋光色散谱2.圆二色谱在生物圆二色谱在生物,医学医学 研究中的应用研究中的应用产生产生,特征特征,XY线线线线偏偏偏偏振振振振光光光光XYE Ex x=E0 cos tEyEy=E0 cos (t- /2)E Ex x2 2 + +Ey2 =E02E Ex x=E0 cos tEyEy=E0 cos (t+ /2)XY频率相同,方向相反振幅相同,相位相同线线偏偏振振光光参照面频率相同,方向相反振幅相同、相位不同线线偏偏振振光光2参照面1 yyxxRL椭椭圆圆偏偏振振光光频率相同,方向相反振幅不同、相位不同参照面21 yyx
2、xRL = 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频频率率 振幅 相位 是否旋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光Optically Active SampleChiral Plane(linearly) polarized lightRightandRightandLefthandLefthandcircularlycircularlypolarizedpolarizedlightlightPreferentia
3、l Preferential absorption absorption of left of left hand hand polarized polarized ?振振动方程方程 E=E0 cos (t+0 )Ex=E0 cos (t-L/v)+0 波波动方程方程Ex=Eo cos t-(2 L/)n+0 Or Or L/v=2L n/c0= 2 / Ln圆二色性二色性与振幅与振幅 A= lgI0/I =(1/2,303)lnI0/I =CLBeer-Lanmbert Law =2.303 A/4 (弧度弧度)A=A=AL-L- AR = C LLOptical active object
4、E Ex x= E0 cos t-(2 L /)n +0 ?C: 摩尔浓度摩尔浓度 L : 光程光程 (dm) l l: : deg mol-1 cm-1 or deg cm2 dmol-1 摩尔椭圆率摩尔椭圆率 , l l the molar ellipticity l lMRw MRw l l /100比椭圆率比椭圆率 l l the specific ellipticity平均残基椭圆率平均残基椭圆率 l lMRw the mean residue ellipticity l l Mw l l /100 = /CL旋光色散旋光色散与位相与位相?Optical active object
5、nl nr = ( L /) n (弧度) n = nl - nr =(2 L /)nLE Ex x=E0 cos t-(2 L/)n+0 ORD (optical rotatory dispersion)平均残基旋光度平均残基旋光度 m the mean residue rotationm = MRw/100C : g/mL L : dmMw: molecular weight MRw: the mean residue Mw比旋光度比旋光度 the specific rotation摩尔比旋光度摩尔比旋光度 j j the molar rotationj j Mw/100 l l / /
6、Cw L Plane(linearly) polarized light基本原理基本原理( principle )RightandRightandLefthandLefthandcircularlycircularlypolarizedpolarizedlightlightOptically Active SamplePreferential Preferential absorption absorption of left of left hand hand polarized polarized ChiralE Ex x=E0x cos t-(2 l /)n+0 Light nl , n
7、r yyxxRL l = f () = f ()ORDCDKronig-Kramerstransitionrelationship between CD and ORD Cotton effect Cotton effect0e00+-+-Positive cottoneffectNegative cottoneffectInstrumenthttp:/www-structure.llnl.gov/CD applicationSecondary structure of macromolecule-helix -helix: 19x nm (+) 208nm, 222nm (-) x E-30
8、200210220 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 (-) 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
9、（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
10、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,补加蔬菜补加蔬菜烟草花叶病毒侵染膜蛋白烟草花叶病毒侵染膜蛋白引自引自:吉尚戎等吉尚戎等生物物理学生物物理学报,2004,20Unfolding/folding216nm222nmConformational varies190200210220230240250260-1.5-1.0-0.50.00.51.01.52.0 a5 in 5 % SDS/PBS a5 in 50 % TFE/PBS a5
11、 in pH7 a5 in 50 % TFE/H2O a5 in H2OX1E-4 deg.cm2/dmol-1wavelength(nm)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. The final S
12、DS 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 rich to beta
13、-sheet richPrPc PrPscScout Near UV spectra: to investigate the tertiary structure振动圆二色谱振动圆二色谱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.
14、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 concentration needs to be adjusted to produce the best data. Changing this has a profound effect on the data, so
15、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 aggregation via Dynamic Light Scattering (DNA repair enzymes are an especially good example o
16、f 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.参考文献参考文献1.生物物理学生物物理学生物物理学生物物理学赵南明，周海梦，高教出版社施普林格出版社赵南明，周海梦，高教出版社施普林格出版社2000,3302.园园园园二二二二色色色色性性性性和和和和旋旋旋旋光光光光色色色色散散散散在在在在分分分分子子子子生生生生物物
17、物物学学学学中中中中的的的的应应应应用用用用鲁鲁子子贤贤，崔崔涛涛，施施庆庆洛洛，科科学学出出版社版社19873. 3.Circular Circular DichroismDichroism and and Linear Linear DichroismDichroism, Alison Rodger and Bengt Nordn, Oxford University Press 19974.分析仪器手册分析仪器手册分析仪器手册分析仪器手册朱良漪，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, p 2475.Circular Dichroism and O
18、ptical Rotary Dispersion of Proteins and Polypeptides, A.J.Alder, N.J.Greenfield and G.D.Fasman, Meth. Enzymology ,27, 675 (1973). 6.Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation, N.Greenfield and G.D.Fasman, Biochemistry, 8 (10), 4108 (1996) 7.C.R.Cantor and P.R.Sch
19、immel, Biophysical Chemistry, Vol.2, Chapter 8 (1980) 8.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) 9.Protein Secondary Structure and Circular Dichroism : A Practical Guide, W.C.Johnson, PROTEINS,
20、 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生的静生的静电场中中时，二者，二者间的的相互作用称相互作用称为电荷荷-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水化水化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生的静生的静电场中中时，二者，二者间的相互作用的相互作用称称为偶极偶极-偶极相互作用，其相互作用能和距离的三次偶极相互作用，其相互作用能和距离的三次方成反比。
21、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 “Adhension”，应改为“Adhesion”P114第13行 “viscsiometer”，应改为“viscosimeter”
22、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“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“pho
23、toxdation”，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 is looked at? What wavelength range does it absorb in?3.You are measuring the CD spectrum of a pro
24、tein using a 1mm path length cell. You find that the signal is weak because the concentration of the protein is lower than you were initially told. How could you overcome this problem and what do you have to be careful of.4.Does the CD spectrum of a protein give most information about the (i) primar
25、y structure, (ii) secondary structure or (iii) tertiary structure?5.Draw the CD spectrum of an -helix. Remember to label the axes and mark important points with their values.6.Why is nitrogen gas used in CD instruments?7.Does CD give information about where the helices are in a protein?8.You wish to study the thermal stability of a protein with a single buried tryptophan residue. What methods would you consider using and why?9.Membrane proteins are difficult to study by CD. True or false? Why?思考问题思考问题思考问题思考问题
