Use of thiol-disulfide exchange method to study transmembrane peptide association in membrane environments

Methods Mol Biol. 2013:1063:3-18. doi: 10.1007/978-1-62703-583-5_1.

Abstract

The development of methods for reversibly folding membrane proteins in a two-state manner remains a considerable challenge for studies of membrane protein stability. In recent years, a variety of techniques have been established and studies of membrane protein folding thermodynamics in the native bilayer environments have become feasible. Here we present the thiol-disulfide exchange method, a promising experimental approach for investigating the thermodynamics of transmembrane (TM) helix-helix association in membrane-mimicking environments. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. This experimental method provides a broadly applicable tool for thermodynamic studies of folding, oligomerization, and helix-helix interactions of membrane proteins.

MeSH terms

  • Cell Membrane / metabolism*
  • Chromatography, High Pressure Liquid
  • Chromatography, Reverse-Phase
  • Detergents / chemistry
  • Disulfides / chemistry*
  • Hydrogen-Ion Concentration
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism*
  • Models, Molecular
  • Oxidation-Reduction
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protein Interaction Mapping / methods*
  • Protein Multimerization
  • Protein Structure, Secondary
  • Sulfhydryl Compounds / chemistry*

Substances

  • Detergents
  • Disulfides
  • Membrane Proteins
  • Peptides
  • Sulfhydryl Compounds