The aggregation state of rhodanese during folding influences the ability of GroEL to assist reactivation

The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents lik e glycerol or low temperature, e.g. refolding at 10 degreesC, successfully retard the off-pathway formation of large inactive aggregates, but the proc ess does not yield 100% active enzyme. These data suggest that mis-folded s pecies are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer t imes before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large agg regates once they are formed, but it could capture some folding intermediat es and activate them, even though they are not capable of forming active en zyme if left to spontaneous refolding. The interaction between GroEL and rh odanese substantially but not completely inhibits intra-protein inactivatio n, which is responsible for incomplete activation during unassisted refoldi ng. Thus, GroEL not only decreases aggregation, but it gives the highest re activation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochi m Biophys. Acta 1382, 120-128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. BioL Chem 275, 63-70).