Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1-2s after mixing. Only in the presence of K(+) that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K(+). Without K(+), a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K(+), a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.
Keywords: DXT; GFP; SAXS; chaperone; chaperonin; conformational change; diffracted X-ray tracking; green fluorescent protein; hyperthermophilic archaea; protein folding; small-angle X-ray scattering.
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