Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways

Evidence that proteins may unfold utilizing complex competing pathways come s from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobe nzoic acid. The kinetics of unfolding of two single cysteine-containing mut ant forms of the small protein barstar, C82A, which contains only Cys40, an d C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing interme diates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other int ermediate, the Cys thiol groups are unreactive. One more long-lived interme diate also needs to be included on the pathway defined by the early interme diate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demons tration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions . (C) 2000 Academic Press.