Abstract
GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.
Highlights
The chaperonin GroEL from Escherichia coli binds numerous proteins in vivo and in vitro and facilitates their folding by providing a space within its unique quaternary structure where protein molecules can safely complete their folding processes [1, 2]
GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 °C
Aldolase was by no means the only protein to display this behavior, and as shown in Fig. 1C, triosephosphate isomerase (TIM), Taka-amylase A, and phosphofructokinase were shown to form stable ternary complexes with GroEL C138W at 25 °C
Summary
Proteins, and other Materials—GroEL C138W was expressed in E. coli JM109/pUCESL cells and purified at 4 °C according to published protocols [14]. Native TIM was denatured for 1 h in 4 M GdnHCl at a protein concentration of 1 mg/ml To initiate refolding, this protein sample was diluted 100-fold in Refolding Buffer B (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 20 mM Mg(CH3COO), and 2 mM dithiothreitol), containing an equimolar concentration of GroEL and GroES oligomers where indicated. Bovine rhodanese (Sigma) was incubated in rhodanese denaturing buffer (37.5 mM Tris-HCl buffer, pH 7.4, containing 6 M GdnHCl and 6 mM dithiothreitol) for 1 h to completely unfold the protein This mixture was diluted with the same denaturing buffer to produce a series of unfolded rhodanese samples with different rhodanese concentrations but a fixed denaturant concentration of 6 M Gdn-HCl. Five l of each unfolded rhodanese sample were rapidly mixed with 500 l of reaction mixture containing 60 nM GroEL C138W tetradecamer and 100 nM wild-type GroES heptamer, which had been preincubated for 10 min at 25 °C. Preliminary experiments showed that in the presence of IPTG, the size of the colonies of E. coli KY1156/pACYCESLC138W after a 24-h incubation at 25 °C was the same as those seen after a 12-h incubation at 37 °C (data not shown)
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