CircularDichroism

1、圓二色譜圓二色譜Circular Dichroism (CD) Application 圓二色光譜儀通過測量生物大分子的圓二色光譜從而得到生物大分子的二級結(jié)構(gòu)。 可應(yīng)用于：蛋白質(zhì)折疊蛋白質(zhì)構(gòu)象研究, DNA/RNA反應(yīng), 酶動力學(xué), 光學(xué)活性物質(zhì)純度測量, 藥物定量分析。天然有機化學(xué)與立體有機化學(xué), 物理化學(xué), 生物化學(xué)與宏觀大分子, 金屬絡(luò)合物, 聚合物化學(xué)等相關(guān)的科學(xué)研究。 構(gòu) 象 確定蛋白質(zhì)構(gòu)象最準確的方法是x-射線晶體衍射，但對結(jié)構(gòu)復(fù)雜、柔性的生物大分子蛋白質(zhì)來說，得到所需的晶體結(jié)構(gòu)較為困難。二維、多維核磁共振技術(shù)能測出溶液狀態(tài)下較小蛋白質(zhì)的構(gòu)象，可是對分子量較大的蛋白質(zhì)的計算處理非常

2、復(fù)雜。 圓二色光譜：研究稀溶液中蛋白質(zhì)構(gòu)象，快速、簡單、較準確 CD 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 spectra of protein in lipid vesicles. We will

3、look at Staphylococcal a-hemolysin as an example主要內(nèi)容主要內(nèi)容CD原理蛋白質(zhì)CD譜CD實驗要點CD原理圓二色性(circular dichroism, CD) 當(dāng)平面偏振光通過具有旋光活性的介質(zhì)時，由于介質(zhì)中同一種旋光活性分子存在手性不同的兩種構(gòu)型，故它們對平面偏振光所分解成的右旋和左旋圓偏振光吸收不同，從而產(chǎn)生圓二色性圓二色性的表示 橢圓度橢圓度 ，摩爾，摩爾橢圓度橢圓度 =2.303(AL AR)/4 = 3298(L - R)3300 (L - R)在蛋白質(zhì)研究中，常用平均殘基摩爾橢圓度圓二色儀原理蛋白質(zhì)的CD譜The peptide b

4、ond is inherently asymmetric & is always optically active蛋白質(zhì)的光學(xué)活性蛋白質(zhì)的CD譜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 蛋白質(zhì)中芳香氨基酸

5、殘基，如色氨酸(Trp)、酪氨酸(Tyr)、苯丙氨酸(Phe)及二硫鍵處于不對稱微環(huán)境時，在近紫外區(qū)250320 nm，表現(xiàn)出CD信號。 Phe殘基: 255、261和268 nm附近；Tyr殘基:277 nm左右；而在279、284和291 nm是Trp殘基的信息；二硫鍵的變化信息反映在整個近紫外CD譜上。 近紫外CD譜可作為一種靈敏的光譜探針，反映Trp、Tyr和Phe及二硫鍵所處微環(huán)境的擾動，能用來研究蛋白質(zhì)三級結(jié)構(gòu)精細變化。Near UV CD spectrum of Lysozyme260270280290300310-200-1000100 nmMain CD features o

6、f protein 2ndary structures - band (nm)+ band (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.

7、Biol.Chem. 259:13253-13261Effect of 50% TFE on a coiled-coilwavelength in nm200210220230240MRE-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

8、 nm200210220230240MRE-35-30-25-20-15-10-50peptide in waterpeptide in 50% TFETFE Can see this by lookingat the effect of trifluoroethanol (TFE) on a coiled-coil similar to GCN4-p1 TFE induces helicity in all peptidesBest fitting procedures use many different proteins for standard spectra There are ma

9、ny 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 helices

10、, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types(Manavalan & Johnson, 1987, Anal. Biochem. 167, 76-85).估算蛋白質(zhì)估算蛋白質(zhì)a a螺旋螺旋含量含量僅適合僅適合a a含量較高的蛋白質(zhì)！含量較高的蛋白質(zhì)！* *YangYang算法算法Limitations of CD secondary structure analysis The simple deconvolution of a CD spectrum into 4 or 5 comp

11、onents which 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

12、 increasing 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.

13、 蛋白的三級結(jié)構(gòu) 1976年，Levitt和Chothia曾在Nature上報道，規(guī)則蛋白質(zhì)的三級結(jié)構(gòu)模型可分為4類 (1) 全型，以僅-螺旋結(jié)構(gòu)為主，其分量大于40 ，而-折疊的分量小于5 (2) 全型，以-折疊這種結(jié)構(gòu)為主，其分量大于40 ，而僅一螺旋的分量小于5 ； (3) +型，螺旋及-疊折分量都大于15 ，這兩種結(jié)構(gòu)在空間上是分離的，且超過60的折疊鏈是反平行排列； (4) /型， -螺旋和B-折疊含量都大于15 ，它們在空間上是相間的，且超過60的折疊鏈平行排列。CD signal of a protein depends on its 2ndary structure chymo

14、trypsin (all b) lysozyme (a + b) triosephosphate isomerase(a/b) myoglobin (all a)從CD譜分析蛋白質(zhì)的結(jié)構(gòu)類型(Venyaminov & Vassilenko)DEF_CLAS.EXE:對全a、 a /b和變性蛋白質(zhì)的準確度為100%，對a + b的準確度為85%，對全b的準確度為75%。 對多肽的判斷較差！CD實驗要點Determination of Protein Concentration精確的方法有： 1 定量氨基酸分析； 2 用縮二脲方法測量多肽骨架濃度 或測氮元素的濃度 ； 3 在完全變性條件下

15、測芳香氨基酸殘基的吸收，來確定蛋白質(zhì)的準確濃度. Not Acceptable: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 w

16、ill absorb radiationHT 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 d

17、irect 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 CH2 during data collection, loosing however a bit the alerting functions of this channel.

18、Bandwidth (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 of

19、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 large a

20、s possible, but not loosing information) 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 da

21、ta points as possible to increase the efficiency of the post run filtering algorithmAccumulation 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 compensates short term rando

22、m 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 values). For long overnight

23、accumulations its essential that room temperature 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.

24、 Its usually difficult to get proper data when absorbance (of sample + solvent) is over 2 O.D.Typical 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 reasonable Stabilizers (Metal ions, etc.): minimum Buffer

25、 Concentration : 5 mM or as low as possible while maintaining protein stability溶劑的吸收溶劑的吸收!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. GlutamateSummary CD is a useful metho