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

University Of Wisconsin Eau Claire, Eau Claire WI

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Abstract

Broadly speaking, our research group studies the interplay between enzyme dynamics and catalysis. This supplemental proposal is developed to acquire Isothermal Titration Calorimeter, which will aid in investigating the effects of molecular crowding on enzyme structure-dynamics- functions. The interior of a cell is extremely crowded, which can potentially impact protein properties such as structure, folding, stability, ligand binding, and enzyme catalysis. The crowding effects could mediate through hard/steric/entropic and/or soft/chemical/enthalpic interactions. Our objective is to investigate the exact mechanism of crowding/confinement and their impacts on substrate binding and catalysis. We are studying the multidomain prolyl-tRNA synthetases (ProRSs), which play a vital role in protein synthesis in all living organisms. Earlier, we demonstrated that domain dynamics is central to ProRSs functions, which are impacted by the crowder-induced shift in the conformational ensemble. Currently, the impact of the “uniform crowding environment” (nonprotein-based crowders) and “structured crowding environment” (protein-based crowders) on conformational dynamics and function of Escherichia coli (Ec) ProRS is being probed. The mechanistic implications of crowding and confinement effects are significant as these enzymes are promising anti-microbial drug targets. The conformational change and crowder-enzyme interactions are being probed by fluorescence spectroscopy, molecular dynamics simulations, and small molecule docking studies, while varying the size, shape, chemical nature, and concentration of the crowders. We are examining the role of molecular crowders on the substrate binding using Saturation Transfer Difference - Nuclear Magnetic Resonance, which requires a large quantity of proteins and kinetics parameters are obtained from enzyme kinetics assays using radiolabeled substrates. The isothermal titration calorimetry (ITC) method is a label-free direct measurement of the heat evolved or absorbed during a binding event between interacting molecules and enzyme-catalyzed reactions and requires less sample. ITC experiments provide the thermodynamic profiles, which would enable us to establish the molecular mechanism of crowding and confinement resulting in enzyme's altered function. A detailed understanding of crowding effects on the properties of multidomain proteins like Ec ProRS could open up new possibilities for protein designing and drug discovery. Moreover, the ITC equipment would enable us to provide hands-on experiences to students in biochemistry and biophysical chemistry courses (~25 students/semester) by designing course-embedded research projects on calorimetry and teaching them the ligand binding thermodynamics in drug discovery.