GROEL-MEDIATED PROTEIN-FOLDING PROCEEDS BY MULTIPLE ROUNDS OF BINDINGAND RELEASE OF NONNATIVE FORMS

The chaperonin GroEL is a ribosome-sized double-ring structure that as sists in folding a diverse set of polypeptides. We have examined the f ate of a polypeptide during a chaperonin-mediated folding reaction. St rikingly, we find that, upon addition of ATP and the cochaperonin GroE S, polypeptide is released rapidly from GroEL in a predominantly nonna tive conformation that can be trapped by mutant forms of GroEL that ar e capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while th e remainder is rebound rapidly by other GroEL molecules. Folding appea rs to occur in an all-or-none manner, as proteolysis and tryptophan fl uorescence indicate that after rebinding, polypeptide has the same str ucture as in the original complex. These observations suggest that Gro EL functions by carrying out multiple rounds of binding aggregation-pr one or kinetically trapped intermediates, maintaining them in an unfol ded state, and releasing them to attempt to fold in solution.