Ban Xiaofeng, Lahiri Pratik, Dhoble Abhishek S, Li Dan, Gu Zhengbiao, Li Caiming, Cheng Li, Hong Yan, Li Zhaofeng, Kaustubh Bhalerao
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, IL-61801, USA.
Comput Struct Biotechnol J. 2019 Jun 26;17:895-903. doi: 10.1016/j.csbj.2019.06.022. eCollection 2019.
The contribution of newly designed salt bridges to protein stabilization remains controversial even today. In order to solve this problem, we investigated salt bridges from two aspects: spatial distribution and evolutionary characteristics of salt bridges. Firstly, we analyzed spatial distribution of salt bridges in proteins, elucidating the basic requirements of forming salt bridges. Then, from an evolutionary point of view, the evolutionary characteristics of salt bridges as well as their neighboring residues were investigated in our study. The results demonstrate that charged residues appear more frequently than other neutral residues at certain positions of sequence even under evolutionary pressure, which are able to form electrostatic interactions that could increase the evolutionary stability of corresponding amino acid regions, enhancing their importance to stability of proteins. As a corollary, we conjectured that the newly designed salt bridges with more contribution to proteins, not only, are qualified spatial distribution of salt bridges, but also, are needed to further increase the evolutionary stability of corresponding amino acid regions. Based on analysis, the 8 mutations were accordingly constructed in the 1,4-α-glucan branching enzyme (EC 2.4.1.18, GBE) from STB02, of which 7 mutations improved thermostability of GBE. The enhanced thermostability of 7 mutations might be a result of additional salt bridges on residue positions that at least one of amino acids positions is conservative, improving their contribution of stabilization to proteins.
即使在今天，新设计的盐桥对蛋白质稳定性的贡献仍然存在争议。为了解决这个问题，我们从两个方面研究了盐桥：盐桥的空间分布和进化特征。首先，我们分析了蛋白质中盐桥的空间分布，阐明了形成盐桥的基本要求。然后，从进化的角度，我们研究了盐桥及其相邻残基的进化特征。结果表明，即使在进化压力下，带电荷的残基在序列的某些位置也比其他中性残基出现得更频繁，它们能够形成静电相互作用，从而增加相应氨基酸区域的进化稳定性，提高它们对蛋白质稳定性的重要性。作为一个推论，我们推测，对蛋白质有更大贡献的新设计盐桥，不仅需要具备合格的盐桥空间分布，还需要进一步提高相应氨基酸区域的进化稳定性。基于分析，我们在来自STB02的1,4-α-葡聚糖分支酶（EC 2.4.1.18，GBE）中相应构建了8个突变，其中7个突变提高了GBE的热稳定性。7个突变体热稳定性的提高可能是由于在残基位置形成了额外的盐桥，其中至少一个氨基酸位置是保守的，从而提高了它们对蛋白质稳定性的贡献。
