Fundamental to our understanding of biological systems is the ability to define interaction surfaces and conformational changes in dynamic protein complexes, yet this objective remains a challenge. Protein footprinting is a powerful solution to this problem. Methods such as hydrogen-deuterium exchange[1] limited proteolysis,[2] and radiolytic cleavage[3] assess changes in protein surface exposure by measuring the susceptibility of the polypeptide backbone to modification. A complementary approach is to use side-chain modification. Because of their unique nucleophilicity, cysteine (Cys) residues at select positions can report

on solvent accessibility and local chemical environment.[4] The reactivity of a Cys residue can be measured to define interaction surfaces at individual amino acid resolution. Moreover, methods have been developed for rapid generation of a library of Cys variants for a protein of interest.