Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Proc Natl Acad Sci U S A. 2009 Sep 8;106(36):15285-90. doi: 10.1073/pnas.0905735106. Epub 2009 Aug 17.

Abstract

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Azides / chemistry*
  • Escherichia coli / enzymology*
  • Flow Cytometry
  • Hydrogen Bonding
  • Kinetics
  • Methionine / metabolism
  • Methionine-tRNA Ligase / genetics*
  • Methionine-tRNA Ligase / metabolism
  • Models, Molecular*
  • Molecular Structure
  • Mutation / genetics
  • Norleucine / analogs & derivatives*
  • Norleucine / chemistry
  • Protein Engineering / methods
  • Staining and Labeling / methods

Substances

  • Azides
  • azidonorleucine
  • Norleucine
  • Methionine
  • Methionine-tRNA Ligase