Kinetically Robust Monomeric Protein from a Hyperthermophile

Abstract

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.