The rates of commitment to renaturation of rhodanese and glutamine synthetase in the presence of the groE chaperonins.

Current models of chaperonin-assisted folding suggest that proteins undergo multiple rounds of binding and release before they are released in a form that is committed to folding to the native state. Using immunoprecipitation techniques, we have determined the rates at which rhodanese and glutamine synthetase (GS) are released from groEL in a form committed to refold to active enzyme. Rhodanese and glutamine synthetase were chosen as substrates because they exhibit different solution requirements for the chaperonin system and they form stable "folding arrested" complexes with groEL. At various times during the groE-dependent renaturations, groEL was rapidly removed from the renaturation mixture by immunoprecipitation and centrifugation (30 s). The conformers that are committed to the native state remained in the supernatant and 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 (t1/2 < 1 min) than for rhodanese (t1/2 = 5 min). In light of previous results, it appears that GS monomers can attain a groE-independent assembly competent conformation after a brief interaction with the chaperonin. In contrast, the renaturation rate for rhodanese 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 chaperonin 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 probably dictated by the hydrophobic nature, number, and lifetimes of the folding intermediates that interact with the chaperonins.