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Bacterial Polysaccharides pp 203–213Cite as

Sulfhydryl Labeling as a Tool to Investigate the Topology of Membrane Proteins Involved in Lipopolysaccharide Biosynthesis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1954))

Abstract

Establishing the topology of membrane proteins, especially when their tridimensional structures are unavailable, is critical to identify functional regions, delimit the protein orientation in the membrane, the number of transmembrane segments, and the position of critical amino acids (whether exposed to the solvent or embedded in the lipid bilayer). Elucidating the topology of bacterial integral membrane proteins typically involves the construction of deletion-fusions whereby regions of the protein are fused to reporters. Although these methods have several advantages, they are also artifact prone. In contrast, methods based on single amino acid substitutions preserve the native protein intact. We describe here an assay to analyze the topology of membrane proteins involved in the biogenesis of bacterial glycoconjugates, which is based on the accessibility of cysteine substitutions at various places in the protein under in vivo and in vitro conditions. Cysteine residues are detected with polyethylene glycol-maleimide (PEG-Mal). This procedure can be applied to crude bacterial cell extracts and does not require protein purification.

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Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research and Cystic Fibrosis Canada to M.A.V. A.F. was supported by a Doctoral Research Scholarship from the Department of Employment and Learning, Northern Ireland.

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Author notes

  1. Faviola Tavares-Carreón and Xiang Ruan contributed equally to this work.

Authors and Affiliations

  1. Centre for Infection and Immunity, Queen’s University Belfast, Belfast, UK

    Amy Ford & Miguel A. Valvano

  2. Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

    Faviola Tavares-Carreón, Xiang Ruan & Miguel A. Valvano

Authors
  1. Faviola Tavares-Carreón
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  2. Xiang Ruan
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  3. Amy Ford
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  4. Miguel A. Valvano
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Corresponding author

Correspondence to Miguel A. Valvano .

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Editors and Affiliations

  1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Inka Brockhausen

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Tavares-Carreón, F., Ruan, X., Ford, A., Valvano, M.A. (2019). Sulfhydryl Labeling as a Tool to Investigate the Topology of Membrane Proteins Involved in Lipopolysaccharide Biosynthesis. In: Brockhausen, I. (eds) Bacterial Polysaccharides. Methods in Molecular Biology, vol 1954. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9154-9_16

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  • DOI: https://doi.org/10.1007/978-1-4939-9154-9_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9153-2

  • Online ISBN: 978-1-4939-9154-9

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