The role of disulfide bond formation in the conformational folding kinetics of denatured/reduced lysozyme

Abstract

Oxidative folding is of vital importance for producing therapeutic proteins in bacteria via recombinant DNA technology since disulfide bonds exist in most pharmaceutical proteins. Although oxidative protein folding has been extensively investigated in vitro, little is explored concerning the role of disulfide formation to protein conformational folding rate. The effects of oxidized (GSSG)/reduced (GSH) glutathione and pH on the conformational folding kinetics of denatured/reduced lysozyme have been studied herein by fluorescence and circular dichroism. It is found that 83% tryptophan residue burial requires disulfide formation, and increasing GSSG concentration greatly accelerates the tertiary structure formation. The fast phase folding rate constant ( k 1) is linearly related to GSSG concentration, indicating the rate-limiting role of mixed-disulfide formation. Moreover, k 1 = 0.006(±0.001) s −1 is likely to be a critical value for judging the determinant of the slow phase folding rate ( k 2), namely, k 2 is controlled by disulfide formation rate only at k 1 < 0.006 s −1. These findings have elucidated the determinants of different folding stages and thus may be beneficial for more efficient control of the oxidative folding of proteins.