Dynamic redox environment-intensified disulfide bond shuffling for protein refolding in vitro: molecular simulation and experimental validation.

One challenge in protein refolding is to dissociate the non-native disulfide bonds and promote the formation of native ones. In this study, we present a coarse-grained off-lattice model protein containing disulfide bonds and simulate disulfide bond shuffling during the folding of this model protein. Introduction of disulfide bonds in the model protein led to enhanced conformational stability but reduced foldability in comparison to counterpart protein without disulfide bonds. The folding trajectory suggested that the model protein retained the two-step folding mechanism in terms of hydrophobic collapse and structural rearrangement. The disulfide bonds located in the hydrophobic core were formed before the collapsing step, while the bonds located on the protein surface were formed during the rearrangement step. While a reductive environment at the initial stage of folding favored the formation of native disulfide bonds in the hydrophobic core, an oxidative environment at a later stage of folding was required for the formation of disulfide bonds at protein surface. Appling a dynamic redox environment, that is, one that changes from reductive to oxidative, intensified disulfide bond shuffling and thus resulted in improved recovery of the native conformation. The above-mentioned simulation was experimentally validated by refolding hen-egg lysozyme at different urea concentrations and oxidized glutathione/reduced glutathione (GSSG/GSH) ratios, and an optimal redox environment, in terms of the GSSG to GSH ratio, was identified. The implementation of a dynamic redox environment by tuning the GSSG/GSH ratio further improved the refolding yield of lysozyme, as predicted by molecular simulation.