Site-Directed Spin Labeling of Proteins

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Abstract

Site-directed spin labeling (SDSL) has emerged as a powerful approach to study structure and dynamics of proteins that are not readily amenable to X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy (13). SDSL involves the site-specific labeling of proteins with spin probes and the use of electron paramagnetic resonance (EPR) spectroscopy for analysis of the labeled proteins. Spin labeling is typically accomplished by cysteine-substitution mutagenesis followed by reaction with a sulfhydryl-specific nitroxide reagent (4). The reagent most widely used is methanethiosulfonate spin label I (5), which generates the nitroxide side chain designated R1, as shown in Fig-1. Other spin label reagents are also used for specific purposes (6), but examples in this chapter make use of R1 exclusively.

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Fig. 1.

Reaction of the methanethiosulfonate spin label (I) with a cysteine residue to generate the side chain R1.