Center for Computational Natural Sciences and Bioinformatics (CCNSB), International Institute of Information Technology Hyderabad (IIIT-H) , Gachibowli, Hyderabad, 500032, India.
J Phys Chem B. 2015 Jan 15;119(2):392-409. doi: 10.1021/jp5079554. Epub 2015 Jan 2.
Improving the thermostability of industrial enzymes is an important protein engineering challenge. Point mutations, induced to increase thermostability, affect the structure and dynamics of the target protein in several ways and thus can also affect its activity. There appears to be no general rules for improving the thermostabilty of enzymes without adversely affecting their enzymatic activity. We report MD simulations, of wild type Bacillus subtilis lipase (WT) and its six progressively thermostable mutants (2M, 3M, 4M, 6M, 9M, and 12M), performed at different temperatures, to address this issue. Less thermostable mutants (LTMs), 2M to 6M, show WT-like dynamics at all simulation temperatures. However, the two more thermostable mutants (MTMs) show the required flexibility at appropriate temperature ranges and maintain conformational stability at high temperature. They show a deep and rugged free-energy landscape, confining them within a near-native conformational space by conserving noncovalent interactions, and thus protecting them from possible aggregation. In contrast, the LTMs having marginally higher thermostabilities than WT show greater probabilities of accessing non-native conformations, which, due to aggregation, have reduced possibilities of reverting to their respective native states under refolding conditions. Our analysis indicates the possibility of nonadditive effects of point mutations on the conformational stability of LTMs.
提高工业酶的热稳定性是蛋白质工程的一个重要挑战。为了提高热稳定性而诱导的点突变会以多种方式影响目标蛋白的结构和动力学，因此也可能影响其活性。似乎没有不影响酶活性而提高酶热稳定性的一般规则。我们报告了野生型枯草芽孢杆菌脂肪酶（WT）及其六个逐渐耐热突变体（2M、3M、4M、6M、9M 和 12M）的 MD 模拟，在不同温度下进行，以解决这个问题。较不热稳定的突变体（LTMs），2M 至 6M，在所有模拟温度下都表现出 WT 样的动力学。然而，两个更耐热的突变体（MTMs）在适当的温度范围内表现出所需的灵活性，并在高温下保持构象稳定性。它们表现出深邃而崎岖的自由能景观，通过保留非共价相互作用将它们限制在近天然构象空间内，从而防止它们可能发生聚集。相比之下，比 WT 具有略高热稳定性的 LTMs 具有更大的可能性进入非天然构象，由于聚集，在复性条件下它们恢复到各自天然状态的可能性降低。我们的分析表明，点突变对 LTMs 构象稳定性的非加性效应的可能性。
