Construction and characterization of the hetero-oligomer of the group II chaperonin from the hyperthermophilic archaeon, Thermococcus sp. strain KS-1

The hyperthermophilic archaeon Thermococcus sp. strain KS-1 (T. KS-1) expresses two different chaperonin subunits, α and β, for the folding of its proteins. The composition of the subunits in the hexadecameric double ring changes with temperature. The content of the β subunit significantly increases according to the increase in temperature. The homo-oligomer of the β subunit, Cpnβ, is more thermostable than that of the α subunit, Cpnα. Since Cpnα and Cpnβ also have different protein folding activities and interactions with prefoldin, the hetero-oligomer is thought to exhibit different characteristics according to the content of subunits. The hetero-oligomer of the T. KS-1 chaperonin has not been studied, however, because the α and β subunits form hetero-oligomers of varying compositions when they are expressed simultaneously. In this study, we characterized the T. KS-1 chaperonin hetero-oligomer, Cpnαβ, containing both α and β in the alternate order, which was constructed by the expression of α and β subunits in a coordinated fashion and protease digestion. Cpnαβ protected citrate synthase from thermal aggregation, promoted the folding of acid-denatured GFP in an ATP-dependent manner, and exhibited an ATP-dependent conformational change. The yield of refolded GFP generated by Cpnαβ was almost equivalent to that generated by Cpnβ but lower than that generated by Cpnα. In contrast, Cpnαβ exhibited almost the same level of thermal stability as Cpnα, which was lower than that of Cpnβ. The affinity of Cpnαβ to prefoldin was found to be between those of Cpnα and Cpnβ, as expected.