Anderson Delaney M, Jayanthi Lakshmi P, Gosavi Shachi, Meiering Elizabeth M
Department of Chemistry, University of Waterloo, Waterloo, ON, Canada.
Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
Front Mol Biosci. 2023 Feb 8;10:1021733. doi: 10.3389/fmolb.2023.1021733. eCollection 2023.
Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two β-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.
动力学稳定性被定义为蛋白质解折叠的速率，它对于确定蛋白质在自然界以及广泛的医学和生物技术应用中的功能寿命至关重要。此外，高动力学稳定性通常与对化学和热变性以及蛋白水解降解的高抗性相关。尽管其具有重要意义，但控制动力学稳定性的具体机制在很大程度上仍不为人所知，并且很少有研究涉及动力学稳定性的合理设计。在此，我们描述了一种设计蛋白质动力学稳定性的方法，该方法利用蛋白质的长程有序性、绝对接触序以及模拟的解折叠自由能垒来定量分析和预测解折叠动力学。我们分析了两种β-三叶蛋白：亲环蛋白，一种具有中等稳定性的近似三重对称天然蛋白，以及三叶蛋白，一种设计的具有极高动力学稳定性的三重对称蛋白。定量分析确定了蛋白质疏水核心区域长程相互作用的显著差异，这部分解释了动力学稳定性的差异。将三叶蛋白的核心相互作用替换成亲环蛋白可提高动力学稳定性，预测的和解折叠速率的实验测量值之间吻合度很高。这些结果证明了易于应用的蛋白质拓扑学测量方法在改变动力学稳定性方面的预测能力，并推荐核心工程作为合理设计动力学稳定性的一个易于处理的目标，该目标可能具有广泛的适用性。
