Site-Directed Mutagenesis and Intrinsic Tryptophan Fluorescence Study to Probe the Conformational Change in Escherichia coli Prolyl-tRNA Synthetase (Ec ProRS)

Abstract

Amino-acyl tRNA synthetases (AARSs) belong to a class of an important family of enzymes that are critical for proteins biosynthesis in all living organisms. They catalyze aminoacylation of tRNAs, a key step in protein synthesis. An important member of AARSs is prolyl-tRNA synthetase (ProRS), a multidomain enzyme in which domain-domain communications are essential for function. These intra-domain communications are mediated by coupled-domain motions, which could be affected by any change in the surrounding environments such as increased concentration of metabolites and biomolecules (molecular crowders). To explore the impact of molecular crowding on the coupled-domain dynamics and functions of ProRSs, we are using synthetic crowding agents to mimic the highly crowded intracellular environment. Our initial kinetics and molecular dynamic simulation studies on Escherichia coli (Ec) ProRS have revealed alterations in catalytic function and protein conformation in the presence of molecular crowders. To determine the site of conformational change(s), site-directed mutagenesis and intrinsic fluorescent studies have been performed. Changes in fluorescence emission intensity and wavelength, which indicate conformational change in the presence of various crowding agents, have been monitored for wild-type and mutant variants of Ec ProRS. Herein, we will present the preliminary results of our work.