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 like glycerol or low temperature, e.g. refolding at 10 degrees C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species 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 times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation 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) Biochim. Biophys. Acta 1382, 120--128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63--70).
Bhattacharyya
A M
AM
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Horowitz
P M
PM
D000818:Animals; D002417:Cattle; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism*; D003080:Cold Temperature; D004789:Enzyme Activation / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D012997:Solvents; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000378:metabolism*
D018835:Chaperonin 10; D018834:Chaperonin 60; D011994:Recombinant Proteins; D012997:Solvents; D013884:Thiosulfate Sulfurtransferase; D005990:Glycerol