Post-Translational Control over Metabolic Labeling of Newly Synthesized Proteins

[摘要] A variety of aminoacyl-tRNA synthetases (aaRSs) have had their substrate specificities altered through protein engineering to create mutant aaRSs that can charge tRNA with noncanonical amino acids. While the activities of these mutant aaRSs can be dynamically controlled in cells using conditional promoters, our ability to quickly switch aaRS-mediated metabolic labeling on and off remains limited because we have not yet discovered ways to directly control aaRS activity post-translationally.I have engineered a first generation of ligand-responsive aaRSs by targeting Escherichia coli methionyl-tRNA synthetase (MetRS). A previously developed mutant, L13N/Y260L/H301L MetRS (NLL-MetRS), can charge tRNA with azidonorleucine (Anl), allowing bioorthogonal metabolic labeling.To develop NLL-MetRS switches, I used a combinatorial approach to generate libraries of split MetRS fused to different protein-protein interactions. I screened these libraries for active split MetRS using bacterial complementation and discovered six split MetRS whose fragments cooperatively function when fused to a pair of interacting proteins. I introduced the mutations necessary to change the specificity of MetRS from methionine to Anl and found that bacterial complementation in split MetRS correlates with metabolic labeling in split NLL-MetRS.I examined whether the activity of split NLL-MetRS can be regulated by either fusing the fragments to a pair of proteins whose interaction is stabilized by ligand binding or by fusing the fragments to the termini of a single protein domain that exhibits a ligand-dependent conformational change. When NLL-MetRS fragments were fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR (FRB), metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-FRB complex. Similarly, fusion of MetRS fragments to the termini of the ligand-binding domain of the human estrogen receptor alpha yielded a protein whose metabolic labeling was significantly enhanced in the presence of 4-hydroxytamoxifen. These protein switches are expected to be useful for extending control over metabolic labeling with Anl. Furthermore, this approach can be applied to metabolic labeling with additional non-natural amino acids by extending the combinatorial design strategy to structurally-related aaRS. Ligand-responsive aaRSs are expected to have applications in production of protein biomaterials, proteomic studies, and protein pharmaceutical biosynthesis.

[发布机构] Rice University

[效力级别] acyl