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圆二色谱Circular Dichroism (CD)
Application 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。
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
CD is very useful for looking at membrane proteins Ø Membrane proteins are difficult to study. Ø Crystallography difficult - need to use detergents Often even when structure obtained: Q- is it the same as lipid? Ø CD ideal can do spectra of protein in lipid vesicles. Ø We will look at Staphylococcal -hemolysin as an example
主要内容 Ø CD原理 Ø 蛋白质CD谱 Ø CD实验要点
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．
圆二色性的表示 椭圆度 ，摩尔椭圆度[ ] =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
蛋白质的光学活性 The peptide bond is inherently asymmetric & is always optically active
蛋白质的CD谱 Ø CD spectra in the far UV region (180 nm – 250 nm) probes the secondary structures of proteins. Ø CD spectra in the near UV region (~250 and ~ 350) monitors the side chain tertiary structures of proteins.
Near 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 250～ 320 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。
Near UV CD spectrum of Lysozyme
Main CD features of protein 2 ndary structures - band (nm) + band (nm) α-helix 222 208 192 β-sheet 216 195 β-turn 220 -230 (weak) 180 -190 (strong) 205 polypro II helix 190 210 -230 weak Random coil 200 212
Far UV CD spectra of poly-L-Lys
CD signals for same secondary structure can vary (a bit) with environment Ø But on a coiled-coil breaks down helical Ø Can see this by looking dimer to single helices at the effect of trifluoroethanol (TFE) on a coiled-coil similar to GCN 4 -p 1 Ø TFE induces helicity in all peptides Ø Although 2 ndry structure same CD changes Lau, Taneja and Hodges (1984) J. Biol. Chem. 259: 13253 -13261
Best fitting procedures use many different proteins for standard spectra Ø Ø There are many different algorithms. All rely on using up to 20 CD spectra of proteins of known structure. By mixing these together a fit spectra is obtained for an unknown. For full details see Dichroweb: the online CD analysis tool www. cryst. bbk. ac. uk/cdweb/html/ Ø Can generally get accuracies of 0. 97 for helices, 0. 75 for beta sheet, 0. 50 for turns, and 0. 89 for other structure types (Manavalan & Johnson, 1987, Anal. Biochem. 167, 76 -85).
估算蛋白质 螺旋含量 仅适合 含量较高的蛋白质！ *Yang算法
Limitations of CD secondary structure analysis Ø 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. Ø The 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 αhelix is known to increase with increasing helix length, CD of βsheets are very sensitive to environment & geometry. Ø Far UV curves (>275 nm) can contain contributions from aromatic amino-acids, in practice CD is measured at wavelengths below this. Ø The shapes of far UV CD curves depend on tertiary as well as secondary structure.
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α型，以仅α-螺旋结构为主，其分量大于40％ ，而β折叠的分量小于5％ (2) 全β型，以β-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) α+β型，α螺旋及β-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) α/β型， α-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。
CD signal of a protein depends on its 2 ndary structure —— chymotrypsin (all ) —— lysozyme ( + ) —— triosephosphate isomerase( / ) —— myoglobin (all )
从CD谱分析蛋白质的结构类型 (Venyaminov & Vassilenko)DEF_CLAS. EXE: 对全 、 / 和变性蛋白质的准确度为 100%， 对 + 的准确度为 85%， 对全 的准确度为 75%。 对多肽的判断较差！
Determination 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. Ø Not Acceptable: 1. Bradford Method. 2. Lowry Method. 3. Absorbance at 280 and/or 260 nm.
Nitrogen flushing Flushing the optics with dry nitrogen is a must: Ø Xe lamp has a quartz envelope, so if operated in air it’ll develop a lot of ozone, harmful for the mirrors Ø below 195 nm oxygen will absorb radiation
HT plot Ø The HT plot is very important, since readings above 600650 V mean that not enough light is reaching the detector so a sample dilution or the use of shorter path cell are required. Ø Furthermore the HT plot is in realty a single beam spectra of our sample, since there is a direct relation 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 CH 2 during data collection, loosing however a bit the alerting functions of this channel.
Bandwidth (SBW) selection Ø 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. Ø As a rule SBW should be kept at least 1/10 of the NBW, otherwise the band will be distorted. Ø If NBW is not known a series 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. Ø 2 nm in the far UV region Ø 1 nm in the aromatic region (where fine structures may be present), optimal band-pass (as large as possible, but not loosing information) can be determined after a trial
Number of data point Ø data 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, it’s advisable to collect as many data points as possible to increase the efficiency of the post run filtering algorithm
Accumulation Ø 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. Ø Averaging is very effective since it compensates short term random noise, but it’ll 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 values). Ø For long overnight accumulations it’s essential that room temperature is well kept stable.
Sample concentration and cell pathlength Ø A good suggestion is to run in advance an absorption UV-VIS spectra. Ø CD spectroscopy calls for same requirements as UV-VIS: best S/N is obtained with absorbance level in the range 0. 6 to 1. 2. It’s usually difficult to get proper data when absorbance (of sample + solvent) is over 2 O. D.
Typical Conditions for protein CD Ø Ø Ø Ø Protein Concentration: 0. 2 mg/ml Cell Path Length: 1 mm Volume 350 ml Need very little sample 0. 1 mg Concentration reasonable Stabilizers (Metal ions, etc. ): minimum Buffer Concentration : 5 m. M or as low as possible while maintaining protein stability
Buffer Systems for CD Analyses Ø Acceptable: 1. Potassium Phosphate with KF, K 2 SO 4 or (NH 4)2 SO 4 as the salt. 2. Hepes, 2 m. M. 3. Ammonium acetate, 10 m. M. Ø Avoid: Tris; Na. Cl; Anything optical active, e. g. Glutamate
Summary Ø CD is a useful method for looking at secondary structures of proteins and peptides. Ø CD is based on measuring a very small difference between two large signals must be done carefully Ø the Abs must be reasonable max between ~0. 6 and ~1. 2. Ø Quarts cells path lengths between 0. 0001 cm and 10 cm. 1 cm and 0. 1 cm common Ø have to be careful with buffers TRIS bad - high UV abs Ø Measure cell base line with solvent Ø Then sample with same cell inserted same way around Ø Turbidity kills - filter solutions Ø Everything has to be clean Ø For accurate 2 ndry structure estimation must know concentration of sample
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
圆二色谱在糖类化合物结构分析中的应用 Ø 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； Ø 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。 Ø 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。