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.
Nishikori
Shingo
S
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292.
Shiraki
Kentaro
K
Okanojo
Masahiro
M
Imanaka
Tadayuki
T
Takagi
Masahiro
M
D019840:2-Propanol / Q000737:chemistry; D000438:Alcohols / Q000494:pharmacology; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D007477:Ions; D007700:Kinetics; D000432:Methanol / Q000737:chemistry; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry / Q000494:pharmacology; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology; D013816:Thermodynamics; D013997:Time Factors; D014270:Trifluoroethanol / Q000494:pharmacology
D000438:Alcohols; D003902:Detergents; D007477:Ions; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D019853:O(6)-Methylguanine-DNA Methyltransferase; D019791:Guanidine; D019840:2-Propanol; D000432:Methanol