Genetically Encoded Tetrazoles for Protein Photocrosslinking
Jacinto, Marco Paolo de Vera
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Many cellular processes are mediated by transient protein-protein interactions. Due to their weak interactions, classical methods to determine these interactions that rely on non-covalent associations such as immunoprecipitation and affinity-based methods may not be suitable. Thus, photocrosslinking methods have been used to alleviate this problem due to its ability to form a covalent linkage between interacting proteins and its high spatial and temporal resolution. Here, we describe the first report of a novel genetically encoded N 6 -(2-(1-methyl-1 H -pyrrol-2-yl)-2 H -tetrazole-5-carbonyl)- L -lysine, we coined as N -methylpyrrole-tetrazole lysine (or mPyTK), and its photocrosslinking reaction based on the conjugation reaction of nitrilimines with nucleophiles in proteins. The orthogonal Methanosarcina mazei PylRS/tRNACUA pair has been evolved for site-specific incorporation of mPyTK into proteins in bacterial and mammalian cells. Furthermore, the utility of mPyTK in efficient photocrosslinking of proteins was demonstrated both in vitro (> 50% conversion) and in vivo (> 75%, E. coli cells) by photoirradiation for 15 min at 302 nm. The substrate promiscuity of the evolved aminoacyl-tRNA synthetase, Mm mPyTKRS, was also evaluated by incorporating a series of mono-aryl analogs of mPyTK into sfGFP in E. coli. Currently, we are extending this approach in mammalian cells to identify proteins that interact with growth factor receptor-bound protein 2 (Grb2), an adaptor protein involved in complex formation between proteins and in growth signaling pathways.