Biochemical Studies to Probe the Domain-Domain Communication Pathways in E. coli Prolyl-tRNA Synthetase Heidi Schmit and Sanchita Hati Department of Chemistry, University of Wisconsin-Eau Claire, WI Biochemical Studies to Probe the Domain-Domain Communication Pathways in E. coli Prolyl-tRNA Synthetase Heidi Schmit and Sanchita Hati Department of Chemistry, University of Wisconsin-Eau Claire, WI Abstract Background Conclusions Methods Active-site titrations will be done to quantify the amount of the protein that is active and to find out the approximate catalytic activity of the wild- type and mutants of ProRS. ATP-PP i exchange assays will be performed for the WT and mutant proteins. This assay involves the first step in the two-step reaction, which is reversible, for the charging of tRNA. This assay is done without the presence of tRNA and the PP i is radioactively labeled so when the reactive goes in reverse direction, the ATP that is produced will become radioactively labeled. This gives an indirect approach to measure the activity of the ProRS. Aminoacylation assays will also be performed that will give the actual measurement of the catalytic activity of the wild-type and mutant enzymes. This assay has the presence of tRNA and is the second step of the aminoacylation two step reaction. Kinetic parameters of the WT and mutant enzymes will be compared to determine the impact of mutation on enzyme function. Double mutants will also be prepared and kinetic parameters will be determined. Overall, these assays will allow us to understand the role of each of the computationally predicted pathways in enzyme function. References- (1) Beuning et al. (2001) J. Biol. Chem. 276, (2) Crepin et al. (2006) Structure 14, (3) Hati et al. (2006) J. Biol. Chem. 281, (4) Johnson et al. (2013) Biochemistry, under revision. (5) Sanford et al. (2012) Biochemistry 51, Acknowledgements:  National Institute of Health (Grant number GM085779)  National Science Foundation through TeraGrid Resources  Office of Research & Sponsored Programs at UWEC  Learning and Technology Services at UWEC To further our understanding of the molecular mechanism of domain-domain communications in ProRS. Perform alanine-scanning mutagenesis to examine if all the predicted pathways of site-to-site communication are equally important for the efficient function of Ec ProRS. To explore the role of tRNA Pro in long-range communication in Ec ProRS. Prolyl-tRNA Synthetase Catalyzes the covalent attachment of proline to tRNA Pro in a two- step reaction: Misactivates alanine and possesses editing mechanism to hydrolyze Ala-tRNA Pro [1] Three domains [2]: Substrate-induced conformational change of catalytically important proline-binding loop (PBL) [2]. Overall activation efficiency (k cat /K M ) of the deletion mutant was decreased ~1200-fold relative to the wild-type enzyme [3] Ten of the twelve site-directed mutations were successful and the DNA sequence of all mutants are verified by DNA sequencing. Overexpression of the ten selected mutants has been completed. Currently, two of the mutants have been purified and concentrated and are ready for the last three steps of Scheme 1. Results Objectives Future Directions Pro + ATP + ProRS ⇆ Pro-AMP∙ProRS + PP i Pro-AMP∙ProRS + tRNA Pro → Pro-tRNA Pro + AMP + ProRS Aminoacyl-tRNA synthetases (AARSs) are a family of enzymes that catalyze the covalent attachment of amino acids to their corresponding transfer-RNA. These enzymes play critical roles in protein synthesis and viability. AARSs are comprised of many domains and each domain is responsible for carrying out a specific function for the proper attachment of correct amino acids to tRNAs. Previous research studies have shown that the protein dynamics, especially correlated motion between residue pairs, play an important role in domain-domain communication in these enzymes. Recently, using molecular simulations and bioinformatics, we have traced several potential pathways of residue-residue interactions through which correlated motions could be propagated from one site to another and thereby help coordinate functions of various domains. We are presently probing those computationally determined pathways through experimental mutational studies. We are specifically focused on one enzyme of this family - prolyl-tRNA synthetases (ProRS), which attaches proline on to the tRNA Pro. Currently, site-directed mutagenesis and kinetic studies are being carried out to study the role of specific mutations in the inter-domain communication and catalytic activity of this enzyme. Preliminary results of this study will be presented. Scheme 1 shows the steps that are involved in site-directed mutagenesis and kinetic studies. The blue boxes are the steps completed for ten of the twelve selected mutants or are in the process of being completed. The gold boxes are the future steps to be completed in the coming year. Aminoacyl-tRNA Synthetases in Translation Basic mechanism of tRNA aminoacylation: An amino acid and cognate tRNA are brought together to catalyze the formation of an ester bond in the active site using ATP. The charged tRNA is then transported to the ribosome for transcription into a protein. Catalaytic/Aminoacylation Domain: responsible for selecting the correct amino and catalyzing the attachment to the tRNA Editing Domain: Assures that the correct amino acid is attached to the tRNA Anticodon binding domain: responsible for recognizing the corresponding tRNA Kinetic parameters for amino acid activation by wild-type (WT) and mutants of E. coli ProRS a a Results are the average of 3 trials with the standard deviation indicated [4]. ΔΔG was calculated according to the equation ΔΔG = -RT ln (fold-decrease of k cat /K M ), where R is the gas constant, cal K -1 mol -1 and T is 310 K. ND indicates not detectable under the experimental conditions used. b Data is from reference [5]. Multiple Pathways of Domain-Domain Communication Representation of residue-residue interaction networks between the aminoacylation domain (R450/C443) and the editing domain (K279). The residues chosen for alanine scanning mutagenesis are shown in blue; residues shown in red are known to have considerable impact on enzyme function. T(h) Supernatant 2. Lysis Buffer 3. Wash Buffer mM Imidazole mM Imidazole Scheme 1. We have the following mutants sequenced and overexpressed: A197G, I404A, I414A, L281A, L304A, N232A, S207A, T199A, and V411A. Two mutants (A197G and L304A) have been purified and concentrated. The picture below on the left is an SDS PAGE gel showing overexpression of one of the mutant proteins using 0.1 M IPTG at 37  C. The second SDS PAGE gel on the right shows the final purified enzyme using affinity column chromatography. Each lane shows the progression of the protein purification. The single, prominent band in lane 5 is the successfully purified protein eluted with 100 mM imidazole.