Multivalent binding of nonnative substrate proteins by the chaperonin GroEL

The chaperonin GroEL binds nonnative substrate protein in the central cavit y of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding AT P and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have address ed this using rings with various combinations of wild-type and binding-defe ctive mutant apical domains, enabled by their production as single polypept ides. A wild-type extent of binary complex formation with two stringent sub strate proteins, malate dehydrogenase or Rubisco, required a minimum of thr ee consecutive binding-proficient apical domains. Rhodanese, a less-stringe nt substrate, required only two wild-type domains and was insensitive to th eir arrangement. As a physical correlate, multivalent binding of Rubisco wa s directly observed in an oxidative cross-linking experiment.