In vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; EC 184.108.40.206) is facilitated by the chaperonin proteins cpn60 and cpn10 from Escherichia coli at 37 °C, but the refolding is strongly inhibited at 10 °C. In contrast, the unassisted refolding of rhodanese is efficient at 10 °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 °C or 2) the recovery of active rhodanese in the complete chaperonin system at 25 °C, after first forming a cpn60-rhodanese complex at 10 °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.
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - 1991|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology