Shiraki K, Nishikori S, Fujiwara S, Hashimoto H, Kai Y, Takagi M, Imanaka T
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Eur J Biochem. 2001 Aug;268(15):4144-50. doi: 10.1046/j.1432-1327.2001.02324.x.
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.
为了获取有关热稳定性与其他因素（如热力学参数、热力学稳定性和其他展开条件）之间关系的信息，对来自嗜热古菌柯达嗜热栖热菌KOD1菌株的O(6)-甲基鸟嘌呤-DNA甲基转移酶（Tk-MGMT）及其嗜温对应物大肠杆菌的C端Ada蛋白（Ec-AdaC）的构象稳定性进行了比较。Tk-MGMT在98.6℃时展开，比Ec-AdaC的展开温度高54.8℃。这两种蛋白质的最大自由能（ΔG(max)）不同；Tk-MGMT的值（29.5℃时为42.9 kJ·mol-1）比Ec-AdaC的值（7.4℃时为16.6 kJ·mol-1）高2.6倍。Tk-MGMT的高构象稳定性归因于其焓值比Ec-AdaC高1.6倍。此外，Tk-MGMT的ΔG(max)温度显著更高（高22.1℃）。Tk-MGMT的变性表观热容（ΔC(p)）比Ec-AdaC低0.7倍。这三种协同效应，即增加ΔGmax、使ΔG与温度曲线移动以及降低ΔC(p)，赋予了Tk-MGMT热稳定性。用四种醇和三种变性剂测试的这两种蛋白质的展开曲线表明，在所有研究条件下，Tk-MGMT都比Ec-AdaC具有更高的稳定性。这些结果表明，Tk-MGMT的高稳定性使其除了对热展开具有抗性外，还对化学展开具有抗性。
