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1、圆二色谱圆二色谱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
2、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 proteinsMembrane 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 spectra of protein in lipid vesicles.We will look
3、 at Staphylococcal -hemolysin as an example主要内容主要内容CD原理蛋白质CD谱CD实验要点CD原理圆二色性(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 椭圆度椭圆度 ，摩尔，摩尔椭圆度椭圆度 =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圆二色仪原理蛋白质的CD谱The peptide bond i
4、s inherently asymmetric & is always optically active蛋白质的光学活性蛋白质的CD谱CD spectra in the far UV region (180 nm 250 nm) probes the secondary structures of proteins.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(T
5、rp)、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 LysozymeMain CD features of protein 2ndary structures - band (nm)+ b
6、and (nm)-helix 222208192-sheet216195-turn220-230 (weak)180-190 (strong)205polypro II helix190210-230 weakRandom coil200212Far UV CD spectra of poly-L-LysCD signals for same secondary structure can vary (a bit) with environment Lau, Taneja and Hodges (1984)J.Biol.Chem. 259:13253-13261But on a coiled-
7、coil breaks down helical dimer to single helicesAlthough 2ndry structure sameCD changesCan see this by lookingat the effect of trifluoroethanol (TFE) on a coiled-coil similar to GCN4-p1TFE induces helicity in all peptidesBest fitting procedures use many different proteins for standard spectraThere a
8、re 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 seeDichroweb: the online CD analysis tool www.cryst.bbk.ac.uk/cdweb/html/Can generally get accuracies of0.97 for helice
9、s, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types(Manavalan & Johnson, 1987, Anal. Biochem. 167, 76-85).估算蛋白质估算蛋白质 螺旋螺旋含量含量仅仅适合适合 含量较高的蛋白质！含量较高的蛋白质！*Yang*Yang算法算法Limitations of CD secondary structure analysisThe simple deconvolution of a CD spectrum into 4 or 5 components wh
10、ich do not vary from one protein to another is a gross over-simplification. 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 -helix is known to increase with increasi
11、ng helix length, CD of -sheets are very sensitive to environment & geometry.Far 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. 蛋白的三级结构 1976年，
12、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。CD signal of a protein depends on its 2ndary structure chymotrypsin (all )
13、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！CD实验要点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.Not Acce
14、ptable:1. Bradford Method.2. Lowry Method.3. Absorbance at 280 and/or 260 nm.Nitrogen 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 radiationHT plotThe HT
15、 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 relation between HT and sampl
16、e 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.Bandwidth (SBW) selectionSetting of
17、 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 of fast survey spectra at different SBW w
18、ill 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 region1 nm in the aromatic region (where fine structures may be present), optimal band-pass (as large as possible, but not loosing information)
19、 can be determined after a trialNumber 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 collect as many data points as possible to increase the ef
20、ficiency of the post run filtering algorithmAccumulationanother 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 compensates short term random noise, but itll not compensate long term
21、 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).For long overnight accumulations its essential that room tempe
22、rature is well kept stable.Sample 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 absorbance level in the range 0.6 to 1.2. Its usually difficult to get proper data w
23、hen absorbance (of sample + solvent) is over 2 O.D.Typical Conditions for protein CDProtein Concentration: 0.2 mg/mlCell Path Length: 1 mmVolume 350 ml Need very little sample 0.1 mgConcentration reasonableStabilizers (Metal ions, etc.): minimumBuffer Concentration : 5 mM or as low as possible while
24、 maintaining protein stability溶剂的吸收溶剂的吸收!Buffer Systems for CD AnalysesAcceptable:1. Potassium Phosphate with KF, K2SO4 or (NH4)2SO4 as the salt.2. Hepes, 2mM.3. Ammonium acetate, 10mM.Avoid: Tris; NaCl; Anything optical active, e.g. GlutamateSummaryCD is a useful method for looking at secondary str
25、uctures of proteins and peptides.CD is based on measuring a very small difference between two large signals must be done carefullythe Abs must be reasonable max between 0.6 and 1.2.Quarts cells path lengths between 0.0001 cm and 10 cm. 1cm and 0.1 cm commonhave to be careful with buffers TRIS bad -
26、high UV absMeasure cell base line with solventThen sample with same cell inserted same way aroundTurbidity kills - filter solutions Everything has to be cleanFor accurate 2ndry 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。 THANKS!
