Daviter Tina, Chmel Nikola, Rodger Alison
ISMB Biophysics Centre, Institute of Structural and Molecular Biology, Birkbeck, University of London, London, UK.
Methods Mol Biol. 2013;1008:211-41. doi: 10.1007/978-1-62703-398-5_8.
Circular dichroism (CD) is the difference in absorption of left and right circularly polarized light, usually by a solution containing the molecules of interest. A non-zero signal for solutions is only measured for chiral molecules such as proteins whose mirror image is not superposable on the original molecule. A CD spectrum provides information about the bonds and structures responsible for the chirality. When a small molecule (or ligand) binds to a protein, it acquires an induced CD (ICD) spectrum through chiral perturbation to its structure or electron rearrangements (transitions). The wavelengths of this ICD are determined by the ligand's own absorption spectrum, and the intensity of the ICD spectrum is determined by the strength and geometry of its interaction with the protein. Thus, ICD can be used to probe the binding of ligands to proteins. This chapter contains an outline of how to perform protein CD and ICD experiments, together with some of the issues relating to experimental design and implementation. Addition of a quarter wave plate to a CD spectropolarimeter converts it to a linear dichroism (LD) spectrometer. When protein samples are aligned either in flow (as for fibers or membrane proteins in liposomes) or on surfaces the orientations of ligands with respect to the protein backbone or other subunits can be determined.
圆二色性（CD）是左旋和右旋圆偏振光吸收的差异，通常由含有感兴趣分子的溶液产生。只有手性分子（如蛋白质，其镜像与原始分子不能完全重叠）的溶液才能测量到非零信号。CD光谱提供了有关负责手性的键和结构的信息。当小分子（或配体）与蛋白质结合时，通过对其结构或电子重排（跃迁）的手性扰动获得诱导CD（ICD）光谱。这种ICD的波长由配体自身的吸收光谱决定，ICD光谱的强度由其与蛋白质相互作用的强度和几何结构决定。因此，ICD可用于探测配体与蛋白质的结合。本章概述了如何进行蛋白质CD和ICD实验，以及一些与实验设计和实施相关的问题。在CD分光偏振计上添加一个四分之一波片可将其转换为线性二色性（LD）光谱仪。当蛋白质样品在流动中（如脂质体中的纤维或膜蛋白）或在表面上排列时，可以确定配体相对于蛋白质主链或其他亚基的取向。
