THE RATES OF COMMITMENT RENATURATION OF RHODANESE AND GLUTAMINE-SYNTHETASE IN THE PRESENCE OF THE GROE CHAPERONINS

Current models of chaperonin-assisted folding suggest that proteins un dergo multiple rounds of binding and release before they are released in a form that is committed to folding to the native state. Using immu noprecipitation techniques, we have determined the rates at which rhod anese and glutamine synthetase (GS) are released from groEL in a form committed to refold to active enzyme. Rhodanese and glutamine syntheta se were chosen as substrates because they exhibit different solution r equirements for the chaperonin system and they form stable ''folding a rrested'' complexes with groEL. At various times during the groE-depen dent renaturations, groEL was rapidly removed from the renaturation mi xture by immunoprecipitation and centrifugation (30 s). The conformers that are committed to the native state remained in the supernatant an d were assayed after 1 h. At 25 degrees C, the rate profiles indicate the release and commitment to folding of GS to its native state occurs far earlier (t(1/2) < 1 min) than for rhodanese (t(1/2) = 5 min). In light of previous results, it appears that GS monomers can attain a gr oE-independent assembly competent conformation after a brief interacti on with the chaperonin. In contrast, the renaturation rate for rhodane se with the groE chaperonins mirrored the committed renaturation rates following groEL depletion. This suggests that rhodanese must interact with groEL throughout most of its folding reaction before it acquires a folding competent (groE independent) state. If current models of ch aperonin mechanism are correct, rhodanese undergoes more rebinding and release cycles than does GS. Structurally, the degree of cycling and hence the rate of commitment to folding to the active form are probabl y dictated by the hydrophobic nature, number, and lifetimes of the fol ding intermediates that interact with the chaperonins.