Cysteine modification: Probing channel structure, function and conformational change

Research output: Chapter in Book/Report/Conference proceedingChapter

7 Scopus citations

Abstract

Cysteine substitution has been a powerful tool to investigate the structure and function of proteins. It has been particularly useful for studies of membrane proteins in their native environment, embedded in phospholipid membranes. Among the 20 amino acids, cysteine is uniquely reactive. This reactivity has motivated the synthesis of a wide array of sulfhydryl reactive chemicals. The commercially available array of sulfhydryl reactive reagents has allowed investigators to probe the local steric and electrostatic environment around engineered cysteines and to position fluorescent, paramagnetic and mass probes at specific sites within proteins and for distance measurements between pairs of sites. Probing the reactivity and accessibility of engineered cysteines has been extensively used in Substituted Cysteine Accessibility Method (SCAM) investigations of ion channels, membrane transporters and receptors. These studies have successfully identified the residues lining ion channels, agonist/antagonist and allosteric modulator binding sites, and regions whose conformation changes as proteins transition between different functional states. The thousands of cysteine-substitution mutants reported in the literature demonstrate that, in general, mutation to cysteine is well tolerated. This has allowed systematic studies of residues in transmembrane segments and in other parts of membrane proteins. Finally, by inserting pairs of cysteines and assaying their ability to form disulfide bonds, changes in proximity and mobility relationships between specific positions within a protein can be inferred. Thus, cysteine mutagenesis has provided a wealth of data on the structure of membrane proteins in their functional environment. This data can complement the structural insights obtained from the burgeoning number of crystal structures of detergent solubilized membrane proteins whose functional state is often uncertain. This article will review the use of cysteine mutagenesis to probe structure-function relationships in ion channels focusing mainly on Cysloop receptors.

Original languageEnglish (US)
Title of host publicationNovel Chemical Tools to Study Ion Channel Biology
PublisherSpringer New York
Pages25-54
Number of pages30
ISBN (Print)9781493928453, 9781493928446
DOIs
StatePublished - Sep 17 2015

Keywords

  • Acetylcholine receptor
  • GABAA receptor
  • Membrane transporter
  • Methanethiosulfonate
  • Potassium channels
  • SCAM
  • Sulfhydryl
  • Thiol
  • Thiolate

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Akabas, M. (2015). Cysteine modification: Probing channel structure, function and conformational change. In Novel Chemical Tools to Study Ion Channel Biology (pp. 25-54). Springer New York. https://doi.org/10.1007/978-1-4939-2845-3_3