Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli.
Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.
Chin
Jason W
JW
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Martin
Andrew B
AB
King
David S
DS
Wang
Lei
L
Schultz
Peter G
PG
D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005815:Genetic Code; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008958:Models, Molecular; D011487:Protein Conformation; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000235:genetics*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase