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

点击翻译按钮获取中文摘要

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.