title: Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid.
abstract: 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.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Nobumasa
authors.initials: N
authors.lastname: Hino
authors.forename: Yuko
authors.initials: Y
authors.lastname: Okazaki
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Akiko
authors.initials: A
authors.lastname: Hayashi
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001407:Bacillus / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents; D066246:ErbB Receptors / Q000378:metabolism* / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D008027:Light; D015336:Molecular Probe Techniques; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D003432:Cross-Linking Reagents; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D046915:Penicillin-Binding Proteins; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors