Poster abstracts

Poster number 25 submitted by Brianne Shane

Role of coupled motions in the catalytic activity of E. coli bacterial prolyl-tRNA synthetases

Brianne Shane (Departments of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH, 43210), Bach Cao (Department of Chemistry, University of Wisconsin, Eau Claire, WI, 54702), Kurt Zimmerman (Department of Chemistry, University of Wisconsin, Eau Claire, WI, 54702), Sudeep Bhattacharyya (Department of Chemistry, University of Wisconsin, Eau Claire, WI, 54702), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH, 43210), Sanchita Hati (Department of Chemistry, University of Wisconsin, Eau Claire, WI, 54702)

Abstract:
Aminoacyl-tRNA synthetases play an integral part in translation by maintaining fidelity in protein synthesis. They catalyze a two-step reaction wherein an amino acid is first activated in the presence of ATP to form an aminoacyl-adenylate. Then in the second step the activated amino acid is aminoacylated to the end of its cognate tRNA. These enzymes are dynamically active assemblies in which there exists a direct link between protein structure, dynamics, and function. Internal dynamics are crucial for maintaining a protein’s biological function including substrate binding and catalysis. Computational and biochemical strategies have been employed to investigate how a single mutation of a noncatalytic residue can modify internal protein dymanics. Specifically, we examined how the motion of the catalytically significant proline binding loop (PBL) in E. coli prolyl-tRNA synthetase is affected by fluctuations at a distant site. The PBL is a mobile loop that folds over the proline moiety of the activated adenylate upon binding. Normal mode analysis revealed that the G217- and E218-containing segment is engaged in correlated motion with the PBL region (residues 190-210). Essential dynamics studies suggest that mutation of G217 or E218 to alanine has significant effects on the motion of the PBL and the overall dynamics of the protein. Mutations of these two highly conserved residues resulted in altered activity of the protein. Cognate proline activation was decreased by ~45-fold in the E218A mutant and ~7-fold in the G217A mutant. Aminoacylation rates were also reduced up to 3-fold. The combined molecular dynamics and experimental mutational studies suggest that coupled motions facilitate substrate binding and activation by bacterial prolyl-tRNA synthetases.

Keywords: aminoacyl-tRNA synthetase