Identification of Methionyl-tRNA Synthetase Mutants Seminar

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