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

表征蛋白质动力学和催化功能之间的联系，以改进酶生物催化剂的设计

• 批准号: RGPIN-2022-04368
• 负责人: Doucet, Nicolas
• 金额: $ 4.08万
• 依托单位: Institut national de la recherche scientifique
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743850
• 关键词: Characterizing; link; between; protein; dynamics

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.

酶是自然界中最有效的催化剂。因此，它们正越来越多地在工业环境中使用，主要是作为有害化合物的一种成本效益和环境友好的替代品。然而，设计专门用于特定应用的新的量身定做的酶仍然是一项非常艰巨和耗时的努力，往往会产生效率低下的生物催化剂。这主要归因于缺乏对蛋白质工程如何影响酶的3D结构、催化功能和分子灵活性的了解。现在已经证实，在催化周转的时间尺度上发生的几个协同的分子运动在促进许多系统的功能方面起着重要的作用。然而，我们还没有全面地了解这种原子的灵活性是如何与催化事件或生物功能耦合的，以及同源的结构折叠是否在进化上依赖于动力学来保持分子的活性。此外，氨基酸序列对这种原子尺度的变构信号传递的影响仍然难以捉摸。我的研究计划的总体长期目标是成功预测和开发控制蛋白质动力学的工具，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。为了应对这一关键挑战，我们首先需要识别和表征选定蛋白质系统中与功能相关的构象亚态，此外还需要提供它们如何管理和影响结构和功能的准确描述。在我们在上一个资助周期开发的实验计算工具的基础上，并使用RNase折叠的结构同源作为概念验证，我们将专注于以下短期研究主题。1)我们将描述和描述来自不同物种的apo和holo核糖核酸酶亚家族同源基因的功能促进动力学，这些基因具有不同/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促进功能的动力学来进化新的构象变异，这些构象变异将被用来以变构的方式调节RNase功能。该项目具有很强的创新性，将为不同的学员群体提供世界级的研究培训。我们方法的新奇之处在于将现代的核磁共振-分子动力学工作流程与先进的半理性进化相结合，以发现服从变构控制的遥远的动态位点。通过提供有关利用诱变调节催化功能的线索，我们的研究计划有可能在应用于具有重大工业意义的生物催化剂的酶工程领域取得根本性突破。此外，通过提供有关构象交换在几个酶系统中的功能作用的信息，拟议的研究将为潜在的蛋白质靶标的变构控制和抑制提供有价值的知识。