Characterizing the link between protein dynamics and catalytic function to improve the design of enzyme biocatalysts

酶是自然界中最有效的催化剂。因此，它们越来越多地用于工业环境，主要是作为一种成本效益高、对环境无害的有害化合物替代品。然而，专门用于特定用途的新定制酶的工程设计仍然是一项非常艰巨和耗时的努力，通常会产生低效的生物催化剂。这主要是由于缺乏对蛋白质工程如何影响酶的三维结构、催化功能和分子柔韧性的理解。现在已经确定，在催化周转的时间尺度上发生的几种协调一致的分子运动在许多系统中起着促进功能的重要作用。然而，我们尚未全面了解这种原子柔韧性如何与催化事件或生物功能耦合，以及同源结构折叠是否在进化上依赖于动力学来保持分子活性。此外，氨基酸序列对这种原子尺度变构信号传输的影响仍然是难以捉摸的。我的研究计划的总体长期目标是成功地预测和开发工具来控制蛋白质动力学，并使用这种变构变量来调节蛋白质工程工作流程中的功能。为了解决这一关键挑战，我们首先需要在选定的蛋白质系统中识别和表征功能相关的构象亚态，此外还需要提供它们如何控制和影响结构和功能的精确描述。基于我们在上一个资助周期中开发的实验计算工具，并使用RNase折叠的结构同源物作为概念验证，我们将专注于以下短期研究主题。1)我们将表征和描述来自不同物种的apo和holo RNase亚家族同源物的功能促进动力学，这些同源物表现出不同/相似的生物学和/或催化功能。2)我们将利用在特定亚家族成员中发现的功能促进动力学来进化新的构象变体，这些构象变体将用于变构调节RNase功能。该项目具有很强的创新性，将为不同群体的学员提供世界一流的研究培训。我们方法的新颖之处在于将现代核磁共振成像工作流程与先进的半理性进化相结合，以发现适合变弹性控制的远距离动态位点。通过提供与利用诱变调节催化功能有关的线索，我们的研究项目有可能在酶工程领域取得根本性突破，应用于具有重要工业意义的生物催化剂。此外，通过提供几种酶系统中构象交换的功能作用的信息，拟议的研究将为潜在蛋白质靶点的变构控制和抑制提供有价值的知识。

Enzymes are the most efficient catalysts in nature. As such, they are increasingly being used in industrial settings, primarily as a cost effective, environmentally friendly alternative to harmful chemical compounds. Yet, the engineering of new tailored enzymes dedicated to specific applications remains a very arduous and time-consuming endeavor that often yields inefficient biocatalysts. This is mainly attributed to a lack of understanding of how protein engineering affects the 3D structure, catalytic function, and molecular flexibility of enzymes. It is now established that several concerted molecular motions occurring on the time scale of the catalytic turnover play an important role in promoting function in numerous systems. However, we have yet to comprehensively understand how this atomic flexibility couples to the catalytic event or biological function, and whether homologous structural folds evolutionarily rely on dynamics to preserve molecular activity. Moreover, the effect of the amino acid sequence on the transmission of this atomic-scale allosteric signal remains elusive. The overall long-term objective of my research program is to successfully predict and develop tools to control protein dynamics and use this allosteric variable to modulate function in protein engineering workflows. To tackle this critical challenge, we first need to identify and characterize functionally relevant conformational sub-states in selected protein systems, in addition to provide a precise description of how they govern and influence structure and function. Building on the experimental-computational tools we developed in the previous funding cycle and using structural homologues of the RNase fold as proof-of-concept, we will focus on the following short-term research themes. 1) We will characterize and describe function-promoting dynamics within apo and holo RNase subfamily orthologues from diverse species that exhibit distinct/similar biological and/or catalytic function. 2) We will harness function-promoting dynamics found within specific subfamily members to evolve new conformational variants that will be used to modulate RNase function allosterically. This program has a strong innovative character and will provide world-class research training to a diverse group of trainees. The novelty of our approach lies in the combination of a modern NMR-MD workflow with advanced semi-rational evolution to uncover distant dynamic sites amenable to allosteric control. By providing clues relating to the modulation of catalytic function using mutagenesis, our research program has the potential to lead to fundamental breakthroughs in the field of enzyme engineering applied to biocatalysts of significant industrial relevance. Additionally, by providing information on the functional role of conformational exchange in several enzyme systems, the proposed research will offer valuable knowledge on the allosteric control and inhibition of potential protein targets.