Equilibrium and kinetic stability of a hyperthermophilic protein, O6-methylguanine-DNA methyltransferase under various extreme conditions.

In this work we have studied the equilibrium and kinetic stability of a hyperthermophilic protein, O(6)-methylguanine-DNA methyltransferase (Tk-MGMT), and its mesophilic counterpart AdaC, in various chemical solutions. In an unfolding experiment using guanidine hydrochloride (GdnHCl), the unfolding free-energy change of Tk-MGMT at 30 degrees C was 42.0 kJ mol(-1), and the half time for unfolding was 4.5 x 10(6) s, which is much slower than that of AdaC and representative mesophilic proteins. In unfolding experiments using methanol, ethanol, 2-propanol, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS), Tk-MGMT retained its native structure at high concentrations, despite the fact that these chemical solutions affect protein conformations in a number of different ways. Kinetic studies using TFE and SDS indicate that the unfolding rates of Tk-MGMT in these solutions are slow as in GdnHCl. Further, the results of a mutational experiment suggest that an ion-pair network plays a key role in this slow unfolding. This slow rate of unfolding under extreme conditions is a significant property that distinguishes Tk-MGMT from mesophilic proteins.