Département de biochimie and Center for Green Chemistry and Catalysis (CGCC), Université de Montréal, Montréal, QC, H3T 1J4, Canada.
PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications, Québec, QC, G1V 0A6, Canada.
Sci Rep. 2019 Apr 30;9(1):6656. doi: 10.1038/s41598-019-42866-8.
Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated β-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.
改变蛋白质的运动可能是成功的障碍，也可能是蛋白质工程的一种使能工具。动力学对蛋白质功能的影响通常只在运动时间尺度的一部分范围内报告。如果运动模式在不同的时间尺度上有很大的差异，那么只有通过广泛监测运动，我们才能理解蛋白质动力学对工程功能性蛋白质的影响。我们采用综合方法，结合实验和计算方法学，阐明了由五个相互关联的β-内酰胺酶组成的实验室工程化系统的快速到缓慢时间尺度（ps 到 ms）的蛋白质动力学：两个天然同源物和三个实验室重组变体。快速（ps-ns）和中间（ns-µs）动力学大多是保守的。然而，在天然同源物中，缓慢运动（µs-ms）很少且保守，但在重组体中则很多且广泛分散。尽管如此，经过修饰的缓慢动力学仍然可以在功能上耐受。高分辨率 X 射线结构的晶体学 B 因子在一定程度上预测了保守运动，但不能预测我们在溶液研究中捕获的新的缓慢运动。我们对连续时间尺度上的蛋白质动力学的检查生动地说明了蛋白质工程动力学影响的复杂性，以及工程化酶系统对新的缓慢运动的功能耐受性。
