In this work we have studied the equilibrium and kinetic stability of a hyperthermophilic protein, O⁶-methylguanine-DNA methyltransferase (Tk-MGMT), and its mesophilic counterpart AdaC, in various chemical solutions. In an unfolding experiment using guanidine hydrochloride (GdnHCI), the unfolding free-energy change of Tk-MGMT at 30°C was 42.0 kJ mol^-1, and the half time for unfolding was 4.5×10⁶ 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 GdnHCI. 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.

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