圆二色谱原理与应用.ppt

CD特点 测量的θ非常小 CD测量的为2.32*(AL-AR) 弧度 以样品有圆二信号一定要有紫外吸收但有紫外吸收不一定由远而信号 CD谱在蛋白质研究中的应用 Recommended Methods For determination of globular protein conformation in solution: SELCON, CDNN and K2D. For determination of polypeptide conformation: LINCOMB with a suitable polypeptide set of references. For determining the effects of mutation s, ligands and perturbants on protein structure: LINCOMB. For evaluating the number of folding states giving rise to a set of spectra: The CCA algorithm and SVD. 从CD谱分析准确性 对全?、 ? /?和变性蛋白质的准确度90-100% 对? + ?的准确度为85% 对全?的准确度为75% Applications of CD in structural biology Determination of secondary structure of proteins that cannot be crystallised Investigation of the effect of e.g. drug binding on protein secondary structure Dynamic processes, e.g. protein folding Studies of the effects of environment on protein structure Secondary structure and super-secondary structure of membrane proteins Study of ligand-induced conformational changes Carbohydrate conformation Investigations of protein-protein and protein-nucleic acid interactions Fold recognition CD实验要点 Nitrogen flushing Flushing the optics with dry nitrogen is a must: Xe lamp has a quartz envelope, so if operated in air it’ll develop a lot of ozone, harmful for the mirrors below 195nm oxygen will absorb radiation HT 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 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. Bandwidth (SBW) selection Setting of slits should be as large as possible (to decrease noise level),