Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-Selective Protein Photo-Cross-Linking

• DOI: 10.1021/jacs.7b02615
• Publication Date: 2017-04-19T18:04:08Z
• Authors: Yulin Tian; Marco Paolo Jacinto; Yu Zeng; Zhipeng Yu; Jun Qu; Wenshe R. Liu; Qing Lin
• Keywords: Models, Molecular; 0301 basic medicine; Molecular Structure; Escherichia coli Proteins; Lysine; Tetrazoles; Photochemical Processes; Amino Acyl-tRNA Synthetases; 03 medical and health sciences; Cross-Linking Reagents; Genetic Code; Humans; GRB2 Adaptor Protein

The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein-protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein-protein interactions and their interfaces in living cells.