Identification of Target Disulfide Bonds Using Mechanism-Based Kinetic Trapping Combined with Differential Cysteine Labeling.

The formation and cleavage of disulfide bonds, tightly controlled processes, are critical to maintain protein structures and functions in many physiological and pathological scenarios. Thiol isomerases catalyze the rearrangement of disulfide bonds and therefore are important in the regulation of protein functions. ERp72, a member in the thiol isomerase family, directly activates integrin Mac-1 by cleaving functional disulfide bonds. Using a mechanism-based kinetic trapping mutant of ERp72, stepwise labeling of cysteines by different alkylating agents allows the identification of specific disulfide bonds targeted by ERp72. The method described here may also be applied to other oxidoreductases to map potential target disulfides.