圆二色谱Circula课件

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1、圆二色谱Circula圆二色谱圆二色谱Circular Dichroism (CD) 圆二色谱CirculaApplication 圆二色光谱仪通过测量生物大分子的圆二色光谱从而得到生物大分子的二级结构。 可应用于：蛋白质折叠蛋白质构象研究, DNA/RNA反应, 酶动力学, 光学活性物质纯度测量, 药物定量分析。天然有机化学与立体有机化学, 物理化学, 生物化学与宏观大分子, 金属络合物, 聚合物化学等相关的科学研究。 圆二色谱Circula构 象 确定蛋白质构象最准确的方法是x-射线晶体衍射，但对结构复杂、柔性的生物大分子蛋白质来说，得到所需的晶体结构较为困难。二维、多维核磁共振技术能测出

2、溶液状态下较小蛋白质的构象，可是对分子量较大的蛋白质的计算处理非常复杂。 圆二色光谱：研究稀溶液中蛋白质构象，快速、简单、较准确 圆二色谱CirculaCD is very useful for looking at membrane proteins Membrane proteins are difficult to study. Crystallography difficult - need to use detergentsOften even when structure obtained:Q- is it the same as lipid? CD ideal can do sp

3、ectra of protein in lipid vesicles. We will look at Staphylococcal a-hemolysin as an example圆二色谱Circula主要内容主要内容CD原理蛋白质CD谱CD实验要点圆二色谱CirculaCD原理圆二色谱Circula圆二色性(circular dichroism, CD) 当平面偏振光通过具有旋光活性的介质时，由于介质中同一种旋光活性分子存在手性不同的两种构型，故它们对平面偏振光所分解成的右旋和左旋圆偏振光吸收不同，从而产生圆二色性圆二色谱Circula圆二色性的表示 椭圆度椭圆度 ，摩尔，摩尔椭圆度椭圆

4、度 =2.303(AL AR)/4 = 3298(L - R)3300 (L - R)在蛋白质研究中，常用平均残基摩尔椭圆度圆二色谱Circula圆二色谱Circula圆二色仪原理圆二色谱Circula蛋白质的CD谱圆二色谱CirculaThe peptide bond is inherently asymmetric & is always optically active蛋白质的光学活性圆二色谱Circula蛋白质的CD谱CD spectra in the far UV region (180 nm 250 nm) probes the secondary structures of pr

5、oteins.CD spectra in the near UV region (250 and 350) monitors the side chain tertiary structures of proteins.圆二色谱CirculaNear UV CD spectrum 蛋白质中芳香氨基酸残基，如色氨酸(Trp)、酪氨酸(Tyr)、苯丙氨酸(Phe)及二硫键处于不对称微环境时，在近紫外区250320 nm，表现出CD信号。 Phe残基: 255、261和268 nm附近；Tyr残基:277 nm左右；而在279、284和291 nm是Trp残基的信息；二硫键的变化信息反映在整个近紫外

6、CD谱上。 近紫外CD谱可作为一种灵敏的光谱探针，反映Trp、Tyr和Phe及二硫键所处微环境的扰动，能用来研究蛋白质三级结构精细变化。圆二色谱Circula圆二色谱CirculaNear UV CD spectrum of Lysozyme260270280290300310-200-1000100 nm圆二色谱CirculaMain CD features of protein 2ndary structures - band (nm)+ band (nm)-helix 222208192-sheet216195-turn220-230 (weak)180-190 (strong)205p

7、olypro II helix190210-230 weakRandom coil200212圆二色谱Circula圆二色谱CirculaFar UV CD spectra of poly-L-Lys圆二色谱CirculaCD signals for same secondary structure can vary (a bit) with environment Lau, Taneja and Hodges (1984)J.Biol.Chem. 259:13253-13261Effect of 50% TFE on a coiled-coilwavelength in nm20021022

8、0230240MRE-35-30-25-20-15-10-50TM-36 aqueousTM-36 + TFETFE But on a coiled-coil breaks down helical dimer to single helices Although 2ndry structure sameCD changesEffect of 50% TFE on a monomeric peptidewavelength in nm200210220230240MRE-35-30-25-20-15-10-50peptide in waterpeptide in 50% TFETFE Can

9、see this by lookingat the effect of trifluoroethanol (TFE) on a coiled-coil similar to GCN4-p1 TFE induces helicity in all peptides圆二色谱CirculaBest fitting procedures use many different proteins for standard spectra There are many different algorithms. All rely on using up to 20 CD spectra of protein

10、s of known structure. By mixing these together a fit spectra is obtained for an unknown. For full details seeDichroweb: the online CD analysis tool Can generally get accuracies of0.97 for helices, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types(Manavalan & Johnson, 1987, Anal

11、. Biochem. 167, 76-85).圆二色谱Circula估算蛋白质估算蛋白质a a螺旋螺旋含量含量仅适合仅适合a a含量较高的蛋白质！含量较高的蛋白质！* *YangYang算法算法圆二色谱CirculaLimitations of CD secondary structure analysis The simple deconvolution of a CD spectrum into 4 or 5 components which do not vary from one protein to another is a gross over-simplification. Th

12、e reference CD spectra corresponding to 100% helix, sheet, turn etc are not directly applicable to proteins which contain short sections of the various structures e.g. The CD of an -helix is known to increase with increasing helix length, CD of -sheets are very sensitive to environment & geometry. F

13、ar UV curves (275nm) can contain contributions from aromatic amino-acids, in practice CD is measured at wavelengths below this. The shapes of far UV CD curves depend on tertiary as well as secondary structure. 圆二色谱Circula蛋白的三级结构 1976年，Levitt和Chothia曾在Nature上报道，规则蛋白质的三级结构模型可分为4类 (1) 全型，以仅-螺旋结构为主，其分量大

14、于40 ，而-折叠的分量小于5 (2) 全型，以-折叠这种结构为主，其分量大于40 ，而仅一螺旋的分量小于5 ； (3) +型，螺旋及-叠折分量都大于15 ，这两种结构在空间上是分离的，且超过60的折叠链是反平行排列； (4) /型， -螺旋和B-折叠含量都大于15 ，它们在空间上是相间的，且超过60的折叠链平行排列。圆二色谱CirculaCD signal of a protein depends on its 2ndary structure chymotrypsin (all b) lysozyme (a + b) triosephosphate isomerase(a/b) myogl

15、obin (all a)圆二色谱Circula从CD谱分析蛋白质的结构类型(Venyaminov & Vassilenko)DEF_CLAS.EXE:对全a、 a /b和变性蛋白质的准确度为100%，对a + b的准确度为85%，对全b的准确度为75%。 对多肽的判断较差！圆二色谱CirculaCD实验要点圆二色谱CirculaDetermination of Protein Concentration精确的方法有： 1 定量氨基酸分析； 2 用缩二脲方法测量多肽骨架浓度 或测氮元素的浓度 ； 3 在完全变性条件下测芳香氨基酸残基的吸收，来确定蛋白质的准确浓度. Not Acceptable:

16、1. Bradford Method.2. Lowry Method.3. Absorbance at 280 and/or 260 nm.圆二色谱CirculaNitrogen flushingFlushing the optics with dry nitrogen is a must:Xe lamp has a quartz envelope, so if operated in air itll develop a lot of ozone, harmful for the mirrorsbelow 195nm oxygen will absorb radiation圆二色谱Circu

17、la圆二色谱CirculaHT plot The HT plot is very important, since readings above 600-650V mean that not enough light is reaching the detector so a sample dilution or the use of shorter path cell are required. Furthermore the HT plot is in realty a single beam spectra of our sample, since there is a direct r

18、elation between HT and sample absorbance. By data manipulation HT conversion into absorbance and buffer baseline subtraction is possible. Alternatively single beam absorbance scale can be used already in CH2 during data collection, loosing however a bit the alerting functions of this channel.圆二色谱Cir

19、culaBandwidth (SBW) selection Setting of slits should be as large as possible (to decrease noise level), but compatible to the natural bandwidth (NBW) of the bands to be scanned. As a rule SBW should be kept at least 1/10 of the NBW, otherwise the band will be distorted. If NBW is not known a series

20、 of fast survey spectra at different SBW will help proper selection. Trade in of accuracy versus sensitivity (i.e. the use of larger than theoretical SBW) is occasionally required. 2 nm in the far UV region 1 nm in the aromatic region (where fine structures may be present), optimal band-pass (as lar

21、ge as possible, but not loosing information) can be determined after a trial圆二色谱CirculaNumber of data pointdata pitch, i.e. number of data points per nm, will not directly influence the noise level. However if post run further data processing will be applied to reduce the noise, its advisable to col

22、lect as many data points as possible to increase the efficiency of the post run filtering algorithm圆二色谱CirculaAccumulation another way to improve S/N is to average more spectra. Here too the S/N will improve with the square root of the number of accumulations. Averaging is very effective since it co

23、mpensates short term random noise, but itll not compensate long term drifts (mainly of thermal origin). So if long accumulations are used we recommend a suitable long warm-up of the system and/or the use of a sample alternator (to collect sequentially sample and blank and average their subtracted va

24、lues). For long overnight accumulations its essential that room temperature is well kept stable.圆二色谱CirculaSample concentration and cell pathlengthA good suggestion is to run in advance an absorption UV-VIS spectra.CD spectroscopy calls for same requirements as UV-VIS: best S/N is obtained with abso

25、rbance level in the range 0.6 to 1.2. Its usually difficult to get proper data when absorbance (of sample + solvent) is over 2 O.D.圆二色谱CirculaTypical Conditions for protein CD Protein Concentration: 0.2 mg/ml Cell Path Length: 1 mm Volume 350 ml Need very little sample 0.1 mg Concentration reasonabl

26、e Stabilizers (Metal ions, etc.): minimum Buffer Concentration : 5 mM or as low as possible while maintaining protein stability圆二色谱Circula溶剂的吸收溶剂的吸收!圆二色谱Circula圆二色谱CirculaBuffer Systems for CD AnalysesAcceptable:1. Potassium Phosphate with KF, K2SO4 or (NH4)2SO4 as the salt.2. Hepes, 2mM.3. Ammonium

27、 acetate, 10mM.Avoid: Tris; NaCl; Anything optical active, e.g. Glutamate圆二色谱CirculaSummary CD is a useful method for looking at secondary structures of proteins and peptides. CD is based on measuring a very small difference between two large signals must be done carefully the Abs must be reasonable

28、 max between 0.6 and 1.2. Quarts cells path lengths between 0.0001 cm and 10 cm. 1cm and 0.1 cm common have to be careful with buffers TRIS bad - high UV abs Measure cell base line with solvent Then sample with same cell inserted same way around Turbidity kills - filter solutions Everything has to b

29、e clean For accurate 2ndry structure estimation must know concentration of sample 圆二色谱Circula核酸的CD信息 B-Z? Or Z-B？建议浓度：在吸光值0.2-0.8时浓度的5-10倍 圆二色谱Circula圆二色谱在糖类化合物结构分析中的应用 碳水化合物的结构决定CD的强度和形状，而从CD获得的构象信息的多少取决于样品结构的复杂程度； 对那些具有较好重复系列的糖类化合物而言，CD能提供更为可靠的空间结构信息。 对那些结构比较复杂的糖类来说，即使不能直接测定糖类化合物的绝对构象，利用一些经验规则，例如，CD可以用来推断糖类是否具有gt构象，而且CD可以作为探针来测定糖类化合物构象的变化，如胶体-溶液或无序-有序的转变过程。圆二色谱Circula THANKS!