Combining Fragmentation of an Amino Acyl tRNA Synthetase with Fusion to Interacting Proteins to Increase Control Over Protein Metabolic Labeling

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Protein synthesis and degradation rates change during development and disease onset, but it remains difficult to measure these changes in multicellular organisms because we lack sufficient spatial and temporal control over proteomic measurements. One method of resolving this issue is to use a modified methionyl tRNA synthetase (MetRS) to regulate noncanonical amino acid tagging of newly synthesized protein with azidonorleucine (Anl). Noncanonical amino acids with azide groups (e.g., Anl) are particularly useful for proteomics because bioorthogonal azides allow for selective “click” chemistry for imaging, enrichment, and sequencing. Anl labeling has been previously accomplished using E. coli MetRS (EcMetRS) harboring NLL (L13N/Y260L/H301L) active site mutations that direct substrate specificity from methionine to Anl. To expand control of this genetically encoded tool for metabolic protein labeling, we built libraries of fragmented EcMetRS that encode all possible fragmented EcMetRS as fusions to different pairs of interacting proteins. Selection of these libraries for active MetRS revealed a large number of backbone cleavage sites that can be tolerated without disrupting protein function. Some of these split variants showed dependence on the protein-protein interaction while others showed activity in the absence of an interaction. Ongoing efforts are examining the activities of these split EcMetRS in the presence of NLL mutations, which are required for Anl labeling. These new split NLL-EcMetRS are expected to enable post-translation regulation of metabolic protein labeling with Anl, which will be useful for increasing control over proteomics in complex cellular settings by allowing for regulation of NLL-EcMetRS through a combination of transcriptional and post-translational control. This work was funded by the Houston Area Molecular Biophysics Training Program and the National Science Foundation.