Experimental Kinetic Study to Explore the Impact of Macromolecular Crowding on Structure and Function of Escherichia coli Prolyl–tRNA Synthetase An Nam Hodac and Dr. Sanchita Hati❖ Department of Chemistry ❖University of Wisconsin-Eau Claire ❖Eau Claire, WI 54701 Abstract Aminoacyl-tRNA synthetases (AARSs) are enzymes that catalyze the covalent attachment of amino acids to their cognate tRNA. This reaction is known as aminoacylation of tRNA and is crucial for protein synthesis in all living organisms. These essential enzymes are large proteins, comprised of multiple domains. It has been proposed that the coupled dynamics between various structural elements of these enzymes are responsible for facilitating enzymatic rate enhancement. Unfortunately, previous in vitro studies were limited to dilute solution environments, and were unable to account for the impact of the macromolecular crowding in the cellular environment on these coupled dynamics. We are employing an experimental, non-radioactive enzyme kinetics approach, to probe the impact of macro molecular crowding agents such as sucrose, dextran, and ficoll-70 on the structure, dynamics, and function of Escherichia coli prolyl-tRNA synthetase. The preliminary data of our comparative study in the absence and presence of crowding agents will be presented. Introduction Method Results Prolyl-tRNA Synthetases (ProRSs) Catalyze the covalent attachment of proline to tRNAPro, which is a two-step reaction: Bacterial ProRS occasionally misactivates alanine and cysteine. E. coli ProRS possesses an editing mechanism and a separate domain to hydrolyze Ala-tRNAPro [1, 2]. Catalysis involves a substrate-induced conformational change of the catalytically important proline-binding loop (PBL) [1]. Dynamics of various domains are coupled [3]. Non radioactive Kinetic assay (malachite green) Aminoacylation of tRNA is a two-step reaction: AA + ATP + AARS AARS•AA-AMP + PPi (1) AARS•AA-AMP + tRNA  AARS + AA-tRNA + AMP (2) Malachite green interacts with inorganic phosphate along with molybdate resulting in a green color. This color change can be used to measure the rate of amino acid activation and aminoacylation of ProRSs in the presence of various macromolecular crowding agents. Sucrose H2O Ficoll 70 + ProRS proline ATP -PPi ProRS•Pro-AMP (Aminoacyl-adenylate) Step 1. Activation of the amino acid. Step 2. Amino acid is transferred to 3′-end of tRNA tRNAPro -AMP Pro-tRNAPro 3′ 5′    +ProRS + Proline + 184mg/ml sucrose +ProRS + Proline + 184mg/ml ficoll 70 +ProRS + Proline + H2O Catalysis by approximation! 5.9Å 40Å Hydrodynamic radius of crowders Malachite Green reaction: Malachite Green AA + ATP + AARS AARS•AA-AMP + PPi (1) +ProRS + Proline + H2O +ProRS + Proline + 200mg/ml sucrose +ProRS + Proline + 200mg/ml ficoll 70 +ProRS + Proline + 100 mg/ml ficoll 70, 100 mg/ml sucrose PPi cleavage with PPiase PPiase + PPi + H2O ↔ 2Pi Malachite Green reaction 5.9Å 40Å 5.9Å and 40Å Malachite Green Solution + 2Pi Hydrodynamic radius of crowders Absorbance at 620 nm Dynamic cross-correlations between the C atoms of the PBL (residues 190-220) and residues 19-565 of the WT and the deletion mutant ∆INS. Editing domain Catalytic domain Anticodon binding domain PLB Pi + (NH4)2MoO4 + H+  H3PMo12O40 H3PMo12O40 + HMG2+ + H+  (MG+)(H2PMo12O40) + 2H+ (yellow, max =446 nm) (Green, max =620 nm) H3PMo12O40 http://img.guidechem.com/casimg/54723-94-3.gif General Procedure and Materials With a constant concentration of protein, we first added varying concentrations of sucrose (hydrodynamic radius 5.9Å) and ficoll 70 (hydrodynamic radius 40Å) to simulate the crowded cell environment [7]. To determine the kinetic parameters, we added fixed concentrations of proline and ProRS and took out aliquots at various time points. Experiments performed using concentrations of 1 mM proline and 100 M ProRS. Objective Conclusions Macromolecular crowding Approximately 20-30% of interior cellular environments are occupied by macromolecular crowding agents (in vivo) [4, 5]. Protein binding affinity as well as catalysis are impacted in the presence of macromolecular crowding agents [6, 7]. Most in vitro studies do not take into account the effects of macromolecular crowding agents on enzymatic rate. Ficoll 70 The non-radioactive malachite green assay has been optimized for our protein system. The presence and increasing size of hydrodynamic radii of macromolecules negatively impacts the kinetics and catalytic efficiency of ProRS. Sucrose Future Work Objective Compare the catalytic reaction of E. coli prolyl-tRNA synthetase by determining the kinetic parameters (kcat and KM) of the two-step aminoacylation reaction in the presence and absence of macromolecular crowding agents. Acknowledgements References Continue studying the impacts of other macromolecules like dextran with different hydrodynamic radii. Determine KM and kcat for ProRS under macromolecular crowding conditions by varying the substrate concentration in order to compare the catalytic efficiency. UWEC-Office of Research and Sponsored Programs Chemistry Department of UWEC National Institute of Health Creping et al. (2006) Structure 14, 1511-1525. (2) Beuning et al. (2001) J. Biol. Chem. 276, 30779-30785 (3) Sandford et al. (2012) Biochemsitry 51, 2146-2156 (4) Ellis, R.J. (2001) Curr. Opin. Struct. Biol. 11, 114 -119. (5) Zimmerman et al (1991), J. Mol. Bol. , 222, 599-620. (6) Batra et al. (2009) Biophys. J. 97, 906 – 911. (7) Schultz et al. (1961) J. Gen. Physiol. 44, 1189-1199