Goedken E R, Marqusee S
Department of Molecular and Cell Biology, University of California, 229 Stanley Hall, Berkeley, CA 94720, USA.
J Mol Biol. 2001 Dec 7;314(4):863-71. doi: 10.1006/jmbi.2001.5184.
Escherichia coli RNase HI is a well-characterized model system for protein folding and stability. Controlling protein stability is critical for both natural proteins and for the development of engineered proteins that function under extreme conditions. We have used native-state hydrogen exchange on a variant containing the stabilizing mutation Asp10 to alanine in order to determine its residue-specific stabilities. On average, the DeltaG(unf) value for each residue was increased by 2-3 kcal/mol, resulting in a lower relative population of partially unfolded forms. Though increased in stability by a uniform factor, D10A shows a distribution of stabilities in its secondary structural units that is similar to that of E. coli RNase H, but not the closely related protein from Thermus thermophilus. Hence, the simple mutation used to stabilize the enzyme does not recreate the balance of conformational flexibility evolved in the thermophilic protein.
大肠杆菌核糖核酸酶HI是用于蛋白质折叠和稳定性研究的一个特征明确的模型系统。控制蛋白质稳定性对于天然蛋白质以及在极端条件下起作用的工程蛋白的开发都至关重要。我们对含有将天冬氨酸10突变为丙氨酸这一稳定突变的变体进行了天然态氢交换，以确定其残基特异性稳定性。平均而言，每个残基的ΔG（展开）值增加了2 - 3千卡/摩尔，导致部分未折叠形式的相对丰度降低。虽然稳定性以统一的系数增加，但D10A在其二级结构单元中显示出的稳定性分布与大肠杆菌核糖核酸酶H相似，而与嗜热栖热菌的密切相关蛋白不同。因此，用于稳定该酶的简单突变并未重现嗜热蛋白中进化出的构象灵活性平衡。
