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. Fig. 1.

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