SpringerProtocols: Abstract: Site-Directed Spin Labeling of Proteins: Applications to Diphtheria Toxin

Site-Directed Spin Labeling of Proteins: Applications to Diphtheria Toxin

By: Kyoung Joon Oh², Christian Altenbach³, R. John Collier², Wayne L. Hubbell³

Abstract

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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 (1–3). 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.

Affiliation(s): (2) Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA
(3) Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA

Book Title: Bacterial Toxins: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 145 | Pub. Date: Mar-10-2000 | Page Range: 147-169 | DOI: 10.1385/1-59259-052-7:147

Subject: Infectious Diseases

References

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