Hollien J, Marqusee S
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA.
Biochemistry. 1999 Mar 23;38(12):3831-6. doi: 10.1021/bi982684h.
The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.
嗜热蛋白获得更高热稳定性的机制仍不清楚，因为通常这些蛋白的序列和结构与其嗜温同源物非常相似。为了深入了解热稳定性的基础，我们测定了两种核糖核酸酶H的蛋白质稳定性曲线，描述了其解折叠自由能对温度的依赖性，一种来自嗜温菌大肠杆菌，另一种来自嗜热菌嗜热栖热菌。监测圆二色性信号作为温度和氯化胍浓度的函数，并将所得的解折叠自由能拟合到吉布斯-亥姆霍兹方程，以获得这些蛋白质的一组热力学参数。尽管这些蛋白质的最大稳定性出现在相似的温度下，但嗜热栖热菌核糖核酸酶H的解折叠热容量较低，导致解折叠自由能对温度的依赖性较小，因此热解链温度较高。此外，这些蛋白质在其各自生物体的最佳生长温度下稳定性相似，这表明热力学稳定性和灵活性的平衡对功能很重要。
