Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu.

The thiol-disulfide exchange reaction plays a central role in the formation of disulfide bonds in newly synthesized proteins and is involved in many aspects of cellular metabolism. Because the thiolate form of the cysteine residue is the key reactive species, its electrostatic milieu is thought to play a key role in determining the rates of thiol disulfide exchange reactions. While modest reactivity effects have previously been seen in peptide model studies, here, we show that introduction of positive charges can have dramatic effects on disulfide bond formation on a structurally restricted surface. We have studied properties of vicinal cysteine residues in proteins using a model system based on redox-sensitive yellow fluorescent protein (rxYFP). In this system, the formation of a disulfide bond between two cysteines Cys149 and Cys202 is accompanied by a 2.2-fold decrease in fluorescence. Introduction of positively charged amino acids in the proximity of the two cysteines resulted in an up to 13-fold increase in reactivity toward glutathione disulfide. Determination of the individual pK(a) values of the cysteines showed that the observed increase in reactivity was caused by a decrease in the pK(a) value of Cys149, as well as favorable electrostatic interactions with the negatively charged reagents. The results presented here show that the electrostatic milieu of cysteine thiols in proteins can have substantial effects on the rates of the thiol-disulfide exchange reactions.