Portal de Periódicos da CAPES

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

2001; Wiley; Volume: 268; Issue: 15 Linguagem: Inglês

10.1046/j.1432-1327.2001.02324.x

1432-1033

Kentaro Shiraki, Shingo Nishikori, Shinsuke Fujiwara, Hiroshi Hashimoto, Yasushi Kai, Masahiro Takagi, Tadayuki Imanaka,

Bacterial Genetics and Biotechnology

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 T m = 98.6 °C, which was 54.8 °C higher than the unfolding temperature of Ec ‐AdaC. The maximum free energy (Δ G max ) of the proteins were different; the value of Tk ‐MGMT (42.9 kJ·mol −1 at 29.5 °C) was 2.6 times higher than that of Ec ‐AdaC (16.6 kJ·mol −1 at 7.4 °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 Δ G max temperature of Tk ‐MGMT was considerably higher (by 22.1 °C). The apparent heat capacity of denaturation (Δ C p ) of Tk ‐MGMT was 0.7‐fold lower than that of Ec ‐AdaC. These three synergistic effects, increasing Δ G max , shifted Δ G vs. temperature curve, and low Δ C 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.