Increasing Spatiotemporal Control over Azidonorleucine Protein Labeling within Cells to Enable Higher Resolution Proteomics

As many signaling pathways are interconnected in complex ways, it is difficult to predict the protein production changes accompanying most changes of cellular state, such as during development or disease onset. One method of resolving this issue is to use a modified methionyl tRNA synthetase (MetRS) for bioorthogonal noncanonical amino acid tagging of newly synthesized protein with azidonorleucine (Anl). Use of noncanonical amino acids with azide groups (e.g., Anl) are particularly useful because azides are uncommon within cells, thus allowing for selective “click” chemistry for imaging and sequencing. Anl labeling can be accomplished using E. coli MetRS (EcMetRS) harboring NLL (L13N/Y260L/H301L) active site mutations that direct substrate specificity from Met to Anl. Using a conditional promoter, EcMetRS activity can be spatiotemporally controlled. To expand spatiotemporal control, we built split two‐fragment EcMetRS libraries with various protein‐protein interactions and linkers with or without Tobacco Etch Virus (TEV) specific protease cleavage sites. The libraries were subject to selection and revealed diverse functional split sites across the protein sequence. Some of these split variants showed dependence on the protein‐protein interaction and on presence/absence of TEV. Overall, these findings suggest that incorporation of the NLL mutations in these new EcMetRS variants will allow for novel regulation of metabolic labeling for proteomics through a combination of transcriptional and post‐translational control. This work was funded by the Houston Area Molecular Biophysics Training Program, the Howard Hughes Medical Institute Med into Grad Program, and the Welch Foundation.