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

  • Faviola Tavares-Carreón
  • Xiang Ruan
  • Amy Ford
  • Miguel A. ValvanoEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Glycosyltransferase Sulfhydryl labeling Membrane proteins O antigen Lipid A-core oligosaccharide Substituted cysteine accessibility mutagenesis 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Faviola Tavares-Carreón
    • 2
  • Xiang Ruan
    • 2
  • Amy Ford
    • 1
  • Miguel A. Valvano
    • 1
    • 2
    Email author
  1. 1.Centre for Infection and ImmunityQueen’s University BelfastBelfastUK
  2. 2.Department of Microbiology and ImmunologyUniversity of Western OntarioLondonCanada

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