Programming post-translational control over the metabolic labeling of cellular proteins with a non-canonical amino acid

Transcriptional control can be used to program cells to label proteins with non-canonical amino acids by regulating the expression of orthogonal aminoacyl tRNA synthetases (aaRSs). However, we cannot yet program cells to control labeling in response to aaRS and ligand binding. To identify aaRSs whose activities can be regulated by interactions with ligands, we used a combinatorial approach to discover fragmented variants of Escherichia coli methionyl tRNA synthetase (MetRS) that require fusion to associating proteins for maximal activity. We found that these split proteins could be leveraged to create ligand-dependent MetRS using two approaches. When a pair of MetRS fragments was fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR, and mutations were introduced that direct substrate specificity towards azidonorleucine (Anl), Anl metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-mTOR complex. In addition, fusion of MetRS fragments to the termini of the ligand-binding domain of the estrogen receptor yielded proteins whose Anl metabolic labeling was significantly enhanced when 4-hydroxytamoxifen was added to the growth medium. These findings suggest that split MetRS can be fused to a range of ligand-binding proteins to create aaRS whose metabolic labeling activities depend upon post-translational interactions with ligands.