Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation

Abstract

Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI.

Keywords: additive; disulfide; folding; glutathione; protein disulfide isomerase.