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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 tRNATyr 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.

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Figure 1: Site-specific incorporation of pBpa in the Grb2 SH2 domain.
Figure 2: Analysis of the interactions between Grb2 and the EGF receptor by coimmunoprecipitation.
Figure 3: Photo-cross-linking of Grb2(pBpa111) with the EGF receptor.
Figure 4: Photo-cross-linking of Grb2 variants, containing pBpa at different positions in the SH2 domain, with the EGF receptor.
Figure 5: Photo-cross-linking of Grb2(pBpa111) with endogenous proteins.

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Acknowledgements

This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Correspondence to Shigeyuki Yokoyama.

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Supplementary Fig. 1

Site-specific incorporation of pBpa into Grb2 in CHO cells. (PDF 166 kb)

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Hino, N., Okazaki, Y., Kobayashi, T. et al. Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nat Methods 2, 201–206 (2005). https://doi.org/10.1038/nmeth739

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