J. Biochem, 2004, Vol. 136, No. 4 503-508
© 2004 The Japanese Biochemical Society
BIOCHEMISTRY |
Equilibrium and Kinetic Stability of a Hyperthermophilic Protein, O6-Methylguanine-DNA Methyltransferase under Various Extreme Conditions
1 School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292; and 2 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510
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 (GdnHCl), 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 x 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 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.
* To whom correspondence should be addressed. Phone: +81-761-51-1650. Fax: +81-761-51-1655. E-mail: takagi{at}jaist.ac.jp