Kinetic Studie on the Functional Expression of Chaperonin

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The objective of the present study was to elucidate quantitatively and physicochemically the molecular mechanism of chaperonin functions by means of physical measurements like small-angle X-ray scattering and kinetic measurements like a stopped-flow technique. The following results were obtained.Although the chaperonin GroEL and GroES from Escherichia coli form the GroEL/GroES complex, its stoichiometry, namely, whether they form a 1: 1 molar-ratio bullet-type complex or rather a 1: 2 molar-ratio football-type complex, was not clear. We used the solution X-ray scattering technique to directly observe the structure of the GroEL/GroES complex in solution under a physiological condition. The results have shown that the bullet-type complex is predominant and that the football-type complex is not present in a significant amount.The multi-sigmoidal behavior of the ATP-concentration dependence of the rate constant of the ATP-induced allosteric transition of the chaperonin GroEL has been interpreted in terms of the double-ring structure of GroEL. In the present study, however, we found that a single-ring mutant(SR1) of GroEL exhibited similar multi-sigmoidal behavior of its ATP dependence. This strongly suggests the presence of at least two binding sites for ATP on GroEL. ADP inhibited the ATP-induced allosteric transition of GroEL, and from the dependence of the apparent rate constant of the allosteric transition on ADP, the second ATP-binding site on GroEL was occupied by ADP at 100 μM ADP and 400 μM of ATP. Under the condition but using azido-ADP instead of ADP, we photochemically labeled GroEL. We confirmed the presence of a phosphate group in the photoaffinity labeled GroEL thus obtained using a fluorescent zinc complex, demonstrating the presence of the second ATP-binding site. We are going to identify the location of the second binding site along the amino acid sequence of GroEL.