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Exploring Uniform, Dual, and Dynamic Topologies of Membrane Proteins by Substituted Cysteine Accessibility Method (SCAM™)

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Bacterial Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2715))

Abstract

A described simple and advanced protocol for Substituted Cysteine Accessibility Method as applied to transmembrane (TM) orientation (SCAM™) permits a topology analysis of proteins in their native state and can be universally adapted to any membrane system to either systematically map an uniform or identify and quantify the degree of mixed topology or establish transmembrane assembly dynamics from relatively static experimental data such as endpoint topologies of membrane proteins. In this approach, noncritical individual amino acids that are thought to reside in the putative extracellular or intracellular loops of a membrane protein are replaced one at the time by cysteine residue, and the orientation with respect to the membrane is evaluated by using a pair of membrane-impermeable non-detectable and detectable thiol-reactive labeling reagents. For the most water-exposed cysteine residues in proteins, the thiol pKa lies in the range of 8–9, and formation of cysteinyl thiolate ions is optimum in aqueous rather in a nonpolar environment. These features and the ease of specific chemical modification with thiol reagents are central to SCAM™. Membrane side-specific sulfhydryl labeling allows to discriminate “exposed, protected or dynamic” cysteines strategically “implanted” at desired positions throughout cysteine less target protein template. The strategy described is widely used to map the topology of membrane protein and establish its transmembrane dynamics in intact cells of both diderm (two-membraned) Gram-negative and monoderm (one-membraned) Gram-positive bacteria, cell-derived oriented membrane vesicles, and proteoliposomes.

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Acknowledgments

This work was supported by NIH grant R01GM121493-6, European Union Marie Skłodowska-Curie Grant H2020-MSCA-RISE-2015-690853, and NATO Science for Peace and Security Programme-SPS 98529.

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Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, McGovern Medical School at The University of Texas Health Science Center at Houston, Houston, TX, USA

    Mikhail Bogdanov

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  1. Mikhail Bogdanov
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Correspondence to Mikhail Bogdanov .

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Editors and Affiliations

  1. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ, CNRS, Marseille, France

    Laure Journet

  2. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ, CNRS, Marseille, France

    Eric Cascales

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Bogdanov, M. (2024). Exploring Uniform, Dual, and Dynamic Topologies of Membrane Proteins by Substituted Cysteine Accessibility Method (SCAM™). In: Journet, L., Cascales, E. (eds) Bacterial Secretion Systems . Methods in Molecular Biology, vol 2715. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3445-5_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3445-5_9

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  • Publisher Name: Humana, New York, NY

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