Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid

Nat Methods. 2005 Mar;2(3):201-6. doi: 10.1038/nmeth739. Epub 2005 Feb 17.


We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Adaptor Proteins, Signal Transducing / radiation effects
  • Amino Acid Substitution
  • Amino Acids / genetics
  • Amino Acids / metabolism*
  • Animals
  • Bacillus / genetics
  • Bacillus / metabolism
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Cross-Linking Reagents
  • ErbB Receptors / metabolism*
  • ErbB Receptors / radiation effects
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • GRB2 Adaptor Protein
  • Humans
  • Light
  • Molecular Probe Techniques*
  • Mutagenesis, Site-Directed
  • Penicillin-Binding Proteins / genetics
  • Penicillin-Binding Proteins / metabolism
  • Photochemistry / methods*
  • Protein Engineering / methods
  • Protein Interaction Mapping / methods*
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / metabolism
  • Spectrum Analysis / methods*


  • Adaptor Proteins, Signal Transducing
  • Amino Acids
  • Cross-Linking Reagents
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Penicillin-Binding Proteins
  • Recombinant Fusion Proteins
  • ErbB Receptors