FIGURE 10.
Strategy for incorporation of a nitrotyrosine into a protein of interest using an orthogonal tRNAUAG/nitro-Tyr-tRNA synthetase. The schematic depicts how the orthogonal incorporation of 3-nitrotyrosine site-specifically at amber codons (TAG) into proteins occurs. The tyrosyl-tRNA synthetase from M. jannaschii is mutated at a number of sites based upon structural information, and a library of these mutant tRNA synthetases is screened for their ability to incorporate the 3-nitrotyrosine but not any other natural amino acid. The selected mutated nitro-Tyr-tRNA synthetase then charges the orthogonal tRNACUA with nitrotyrosine, which is then translationally incorporated into the protein of interest site-specifically with high fidelity at the TAG codon. aaRS, aminoacyl-tRNA synthetase.