Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Mol Cell. 2009 Dec 11;36(5):861-71. doi: 10.1016/j.molcel.2009.11.022.
Identifying mutations that stabilize proteins is challenging because most substitutions are destabilizing. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how evolution has formed today's protein sequences. Here we describe a genetic selection that directly links the in vivo stability of proteins to antibiotic resistance. It allows the identification of stabilizing mutations within proteins. The large majority of mutants selected for improved antibiotic resistance are stabilized both thermodynamically and kinetically, indicating that similar principles govern stability in vivo and in vitro. The approach requires no prior structural or functional knowledge and allows selection for stability without a need to maintain function. Mutations that enhance thermodynamic stability of the protein Im7 map overwhelmingly to surface residues involved in binding to colicin E7, showing how the evolutionary pressures that drive Im7-E7 complex formation have compromised the stability of the isolated Im7 protein.
鉴定稳定蛋白质的突变是具有挑战性的，因为大多数取代都是不稳定的。除了具有巨大的实际效用之外，在体内进化蛋白质稳定性的能力还可以表明进化是如何形成当今的蛋白质序列的。在这里，我们描述了一种遗传选择，该选择可将蛋白质的体内稳定性与抗生素抗性直接联系起来。它允许鉴定蛋白质内的稳定突变。选择提高抗生素抗性的大多数突变体在热力学和动力学上均得到稳定，这表明相似的原则在体内和体外控制稳定性。该方法不需要事先的结构或功能知识，并且允许在不需要维持功能的情况下进行稳定性选择。增强蛋白质 Im7 热力学稳定性的突变主要集中在与大肠杆菌素 E7 结合的表面残基上，这表明驱动 Im7-E7 复合物形成的进化压力如何损害了分离的 Im7 蛋白质的稳定性。
