Exploring the Dynamics of Prolyl-tRNA Synthetases: Towards Developing a Screening Method for Species-Specific Inhibitors

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The proposed work is the renewal of our previously funded NIH grant proposal. Our research is focused on the interplay between enzyme dynamics and catalysis. For the past twelve years, we have developed techniques and protocols to explore some pressing issues of enzymology − the precise role of dynamics in catalysis, the evolution of functional dynamics across species, and the impact of molecular crowding on catalysis. We are using prolyl-tRNA synthetase (ProRS) as our model enzyme, which belongs to the superfamily of enzymes called aminoacyl-tRNA synthetases (AARSs). Because of their central role in protein synthesis, AARSs have emerged as attractive targets for anti-infective drug development. Common to all kingdoms of life, the active site of ProRSs from different species bear significant sequence similarity. As a result, drug molecules targeting a pathogenic enzyme's catalytic site could bind to the human counterpart, thus resulting in cell toxicity. Therefore, we are attempting to utilize proteins' intrinsic dynamics for designing and screening species-specific inhibitors for pathogenic ProRSs. Currently, the work is at a juncture, where a thorough investigation using state-of-the-art computational techniques, standardized biochemical protocols, and well-established spectroscopic methods could provide deeper insights into the above-mentioned topics. Herein, we propose a comprehensive study, which blends classical and modern biochemical and biophysical techniques to address these core questions of enzymology. The proposed study will investigate in detail the interplay of electrostatics and dynamics in a quest for the evolutionary origin of the enzyme's catalytic power. Additionally, the proposed study will strive to determine the effects of the crowded cellular milieu on our model enzyme. This will be accomplished by following the molecular details of enzymes' conformational dynamics, substrate recognition, and catalysis in crowded and confined environments. Completion of the proposed work could lead to new possibilities for protein design and drug discovery. In addition to the scientific relevance, the proposed work would provide an outstanding opportunity for our students; participation will yield the development of hands-on laboratory skills while deepening their understanding of these fundamental biophysical concepts. Furthermore, the experience and skills acquired through participation will lead to preparedness for the workforce, as well as opportunities to thrive in graduate and professional schools.