Folding of Disulfide Proteins

Abstract

Unfolding and refolding of disulfide proteins can be investigated by the method of disulfide scrambling which is based on the reversible conversion between the native (N) and scrambled isomers (X). The method of disulfide scrambling presents a number of unique features in elucidation of pathways of protein unfolding and refold- ing. (a) It allows trapping and isolation of diverse intermediates (unfolding and folding) for further structural analysis. (b) It demonstrates that protein denaturation and unfold- ing can be quantified independently. Denaturation is calculated by the conversion of native (N) to non-native X-isomers. Unfolding is measured by the progressive unfold- ing of X-isomers. (c) It shows that folding experiment can be initiated with a structur- ally defined X-isomer possessing the highest free energy among all unfolded X-isomers. (d) It reveals that the energy landscape of conformational heterogeneity (unfolding and refolding) can be illustrated by a diamond-shaped model. At two extreme ends of the energy landscape, the conformational heterogeneity is reduced to minimum