Cooke James A, Brown Louise J
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Methods Mol Biol. 2011;752:73-96. doi: 10.1007/978-1-60327-223-0_6.
Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) offers a powerful method for the structural analysis of protein folds. This method can be used to test and build secondary, tertiary, and quaternary structural models as well as measure protein conformational changes in solution. Insertion of two cysteine residues into the protein backbone using molecular biology methods and the subsequent labeling of the cysteine residues with a paramagnetic spin label enables the technique of EPR to be used as a molecular spectroscopic ruler. EPR measures the dipolar interaction between pairs of paramagnetic spin labels to yield internitroxide distances from which quantitative structural information on a protein fold can then be obtained. Interspin dipolar interaction between two spin labels at less than 25 Å are measured using continuous wave (CW) EPR methods. As for any low-resolution distance methods, the positioning of the spin labels and the number of distance constraints to be measured are dependent on the structural question being asked, thus a pattern approach for using distance sets to decipher structure mapping, including protein folds and conformational changes associated with biological activity, is essential. Practical guidelines and hints for the technique of SDSL-EPR are described in this chapter, including methods for spin labeling the protein backbone, CW-EPR data collection at physiological temperatures and two semiquantitative analysis methods to extract interspin distance information from the CW-EPR spectra.
定点自旋标记电子顺磁共振（SDSL-EPR）为蛋白质折叠的结构分析提供了一种强大的方法。该方法可用于测试和构建二级、三级和四级结构模型，以及测量溶液中蛋白质的构象变化。利用分子生物学方法将两个半胱氨酸残基插入蛋白质主链，并随后用顺磁自旋标记物标记半胱氨酸残基，使得EPR技术能够用作分子光谱尺。EPR测量顺磁自旋标记物对之间的偶极相互作用，以产生氮氧化物间距离，进而可从中获得关于蛋白质折叠的定量结构信息。使用连续波（CW）EPR方法测量两个自旋标记物之间小于25 Å的自旋间偶极相互作用。与任何低分辨率距离方法一样，自旋标记物的定位和要测量的距离约束数量取决于所提出的结构问题，因此，使用距离集来破译结构映射（包括蛋白质折叠和与生物活性相关的构象变化）的模式方法至关重要。本章介绍了SDSL-EPR技术的实用指南和提示，包括蛋白质主链自旋标记的方法、生理温度下的CW-EPR数据收集以及从CW-EPR光谱中提取自旋间距离信息的两种半定量分析方法。
