Xue Zixiao, Quan Shu
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), East China University of Science and Technology, Shanghai 200237, China.
Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Shanghai 200237, China.
ACS Omega. 2023 Jul 13;8(29):25739-25748. doi: 10.1021/acsomega.3c00373. eCollection 2023 Jul 25.
Point mutations can exert beneficial effects on proteins, including stabilization. The stabilizing effects of mutations are typically attributed to changes in free energy and residue interactions. However, these explanations lack detail and physical insights, which hinder the mechanistic study of protein stabilization and prevent accurate computational prediction of stabilizing mutations. Here, we investigate the physical mechanism underlying the enhanced thermostability of a Hygromycin B phosphotransferase mutant, Hph5. We find that the unpredictable mutation A118V induces rotation of F199, allowing it to establish an aromatic-aromatic interaction with W235. In contrast, the predictable mutation T246A acts through static hydrophobic interactions within the protein core. These discoveries were accelerated by a residue-coevolution-based theory, which links mutational effects to stability-associated local structures, providing valuable guidance for mechanistic exploration. The established workflow will benefit the development of accurate stability prediction programs and can be used to mine a protein stability database for undiscovered physical mechanisms.
点突变可以对蛋白质产生有益影响，包括稳定性增强。突变的稳定作用通常归因于自由能和残基相互作用的变化。然而，这些解释缺乏细节和物理见解，这阻碍了对蛋白质稳定性的机制研究，并妨碍了对稳定突变的准确计算预测。在这里，我们研究了潮霉素B磷酸转移酶突变体Hph5热稳定性增强的物理机制。我们发现不可预测的突变A118V诱导F199旋转，使其能够与W235建立芳香-芳香相互作用。相比之下，可预测的突变T246A通过蛋白质核心内的静态疏水相互作用起作用。基于残基协同进化的理论加速了这些发现，该理论将突变效应与稳定性相关的局部结构联系起来，为机制探索提供了有价值的指导。所建立的工作流程将有利于准确稳定性预测程序的开发，并可用于挖掘蛋白质稳定性数据库以寻找未发现的物理机制。
