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.
Shiraki
K
K
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Nishikori
S
S
Fujiwara
S
S
Hashimoto
H
H
Kai
Y
Y
Takagi
M
M
Imanaka
T
T
D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D029968:Escherichia coli Proteins; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D019791:Guanidine