Structural Stability of Oligomeric Chaperonin 10: the Role of Two β-Strands at the N and C Termini in Structural Stabilization

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10 kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding–refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 °C, both cpn10s showed unfolding–refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel β-strands at the N and C termini of this protein. From analyses of the mutantsʹ stabilities, it was revealed that the anti-parallel β-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.