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Bacterial Toxins: Methods and Protocols pp 147–169Cite as

Site-Directed Spin Labeling of Proteins

Applications to Diphtheria Toxin

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 145))

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 (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.

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

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Author information

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA

    Kyoung Joon Oh & R. John Collier

  2. Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA

    Christian Altenbach & Wayne L. Hubbell

Authors
  1. Kyoung Joon Oh
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  2. Christian Altenbach
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  3. R. John Collier
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  4. Wayne L. Hubbell
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Editors and Affiliations

  1. Research Center Borstel, Germany

    Otto Holst

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© 2000 Humana Press Inc.

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Joon Oh, K., Altenbach, C., Collier, R.J., Hubbell, W.L. (2000). Site-Directed Spin Labeling of Proteins. In: Holst, O. (eds) Bacterial Toxins: Methods and Protocols. Methods in Molecular Biology™, vol 145. Humana Press. https://doi.org/10.1385/1-59259-052-7:147

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  • DOI: https://doi.org/10.1385/1-59259-052-7:147

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-604-8

  • Online ISBN: 978-1-59259-052-0

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