Identification of Methionyl-tRNA Synthetase Mutants Seminar <br />

Incorporation of noncanonical amino acids into cellular proteins often requires

engineering new aminoacyl-tRNA synthetase (aaRS) activity into the cell, usually

by modifying natural aaRSs. Although experimental methods, by relying on

mutagenesis and library screening, have identified many successful mutant

aaRS-substrate pairs in recent years, computational approaches have resulted in

only a few successes. Here we compare the results of computational and

experimental screens of an E. coli methionyl-tRNA synthetase (MetRS)

saturation-mutagenesis library for binding (in silico) and activation (in vivo) of

azidonorleucine (Anl). Using a screening strategy that relies on cell-surface

display of reactive amino acid side-chains, we have discovered a set of MetRS

mutants with varying levels of activity towards Anl. Binding energies computed

on models of Anl-bound mutants correlate well with kinetic parameters of Anl

activation measured in vitro. Our results highlight the strengths and weaknesses

of the computational methodology and contribute to our understanding of ligand

recognition by MetRS. Furthermore, mutants discovered here have enabled

proteomic studies on selected cells in complex cell mixtures.

İ. Çağlar Tanrıkulu

Division of Chemistry and Chemical Engineering

California Institute of Technology

FENS - L047 / Thursday Jan 7, 12:40