Intermediates in the chaperonin-assisted refolding of rhodanese are trapped at low temperature and show a small stoichiometry.
In vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; EC 2.8.1.1) is facilitated by the chaperonin proteins cpn60 and cpn10 from Escherichia coli at 37 degrees C, but the refolding is strongly inhibited at 10 degrees C. In contrast, the unassisted refolding of rhodanese is efficient at 10 degrees C, but the refolding efficiency decreases as the temperature is raised. These observations provided two measures of the cpn60-rhodanese complex. Thus, we monitored either 1) the cpn60-dependent inhibition of spontaneous folding at 10 degrees C or 2) the recovery of active rhodanese in the complete chaperonin system at 25 degrees C, after first forming a cpn60-rhodanese complex at 10 degrees C. These procedures minimized the aggregation of interactive folding intermediates that tend to overestimate the apparent number of cpn60 14-mers in determining the stoichiometry of protein-cpn60 14-mer interactions. Both procedures used here gave results that were consistent with there being 1 rhodanese binding site/cpn60 tetradecamer. This stoichiometry is significantly less than might be expected from the fact that cpn60 is composed of 14 identical subunits, and it may indicate that rhodanese interacts with a restricted region that is formed when the cpn60 tetradecamer is assembled. The ability to stabilize chaperonin-protein complexes that can subsequently be reactivated will aid studies of the mode of action of the ubiquitous chaperonin proteins.
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J A
JA
Mendoza
G H
GH
Lorimer
P M
PM
Horowitz
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D018834:Chaperonin 60; D018833:Chaperonins; D006360:Heat-Shock Proteins / Q000737:chemistry; D007700:Kinetics; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013696:Temperature; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry*; D014508:Urea
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011506:Proteins; D000255:Adenosine Triphosphate; D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D018833:Chaperonins