Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart.

Comparison of the conformational stability of an O(6)-methylguanine-DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-MGMT), and its mesophilic counterpart C-terminal Ada protein from Escherichia coli (Ec-AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk-MGMT unfolded at Tm = 98.6 degrees C, which was 54.8 degrees C higher than the unfolding temperature of Ec-AdaC. The maximum free energy (DeltaG(max)) of the proteins were different; the value of Tk-MGMT (42.9 kJ.mol-1 at 29.5 degrees C) was 2.6 times higher than that of Ec-AdaC (16.6 kJ.mol-1 at 7.4 degrees C). The high conformational stability of Tk-MGMT was attributed to a 1.6-fold higher enthalpy value than that of Ec-AdaC. In addition, the DeltaG(max) temperature of Tk-MGMT was considerably higher (by 22.1 degrees C). The apparent heat capacity of denaturation (DeltaC(p)) of Tk-MGMT was 0.7-fold lower than that of Ec-AdaC. These three synergistic effects, increasing DeltaGmax, shifted DeltaG vs. temperature curve, and low DeltaC(p), give Tk-MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk-MGMT possessed higher stability than Ec-AdaC in all conditions studied. These results indicate that the high stability of Tk-MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.