Reversible denaturation of oligomeric human chaperonin 10: Denatured statedepends on chemical denaturant

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have c haracterized denaturation and renaturation of the recombinant human chapero nin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical de naturants urea and guanidine hydrochloride (GuHCl) as well as by heat was m onitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced de naturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under t hese conditions cpn10 heptamers denature without dissociation. There were n o indications of equilibrium intermediates, such as folded monomers, in eit her denaturant. The different cpn10 denatured states observed in high. [GuH Cl] and high [urea] were supported by cross-linking experiments. Thermal de naturation revealed that monomer and heptamer reactions display the same en thalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combin ing chemical denaturation with the dissociation constant in absence of dena turant, shows that dissociated monomers are only marginally stable (3 kJ/mo l). The thermodynamics for co-chaperonin stability appears conserved; there fore, instability of the monomer could be necessary to specify the native h eptameric structure.