Abstract：
As a leading contender in the study of luminescence, nanoluciferase has recently attracted attention and proven effective in a wide variety of research areas. Although numerous attempts have been made to improve activity, there has yet to be a thorough exploration of further possibilities to improve thermostability. In this study, protein engineering in tandem with molecular dynamics simulation at various temperatures (300 K, 400 K, 450 K and 500 K) was used to improve our understanding of nanoluciferase dynamics and identification of factors that could significantly enhance the thermostability. Based on these, three novel mutations have been narrowed down, which were hypothesised to improve thermostability. Root mean square deviation and root mean square fluctuation studies confirmed higher stability of mutant at high temperature. Solvent-accessible surface area and protein unfolding studies revealed a decreased tendency of mutant to unfold at higher temperatures. Further free energy landscape and principal component analysis was adapted to get deeper insights into the thermodynamic and structural behavior of these proteins at elevated temperature. Thus, this study provides a deeper insight into the dynamic factors for thermostability and introduces a novel, enhanced nanoluciferase candidate with potential use in industry.Communicated by Ramaswamy H. Sarma.
摘要：
作为发光研究的主要竞争者，纳米荧光素酶最近引起了人们的注意，并在各种研究领域被证明是有效的。尽管已经进行了许多尝试来改善活动，还有尚未彻底探索进一步的可能性，以提高热稳定性。在这项研究中,蛋白质工程与不同温度下的分子动力学模拟(300K，400K,450K和500K）用于提高我们对纳米荧光素酶动力学的理解以及对能显着增强热稳定性的因素的识别。基于这些,三个新的突变已经缩小了，它们被假设为提高热稳定性。均方根偏差和均方根波动研究证实了突变体在高温下的更高稳定性。溶剂可及的表面积和蛋白质解折叠研究表明，突变体在较高温度下解折叠的趋势降低。进一步的自由能景观和主成分分析进行了调整，以更深入地了解这些蛋白质在高温下的热力学和结构行为。因此，这项研究为热稳定性的动态因素提供了更深入的见解，并介绍了一种新颖的，在工业中具有潜在用途的增强纳米荧光素酶候选物。由RamaswamyH.Sarma沟通。
