A THERMODYNAMIC COUPLING MECHANISM FOR GROEL-MEDIATED UNFOLDING

Chaperonins prevent the aggregation of partially folded or misfolded f orms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exer ts this function by unfolding such intermediates, presumably in a cata lytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant o f RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion , because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroE L is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM -T1 because it binds with high affinity to the unfolded form of the pr otein and thereby shifts the overall equilibrium toward the unfolded s tate. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of t he binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.