Thomas Emily E, Pandey Naresh, Knudsen Sarah, Ball Zachary T, Silberg Jonathan J
Department of Biosciences, ‡Biochemistry and Cell Biology Graduate Program, §Department of Chemistry, ∥Department of Bioengineering, Rice University , Houston, Texas 77005, United States.
ACS Synth Biol. 2017 Aug 18;6(8):1572-1583. doi: 10.1021/acssynbio.7b00100. Epub 2017 May 17.
Transcriptional control can be used to program cells to label proteins with noncanonical 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 (FRB) of mTOR and mutations were introduced that direct substrate specificity toward azidonorleucine (Anl), Anl metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-FRB 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 (4-HT) was added to the growth medium. These findings suggest that split MetRS can be fused to a range of ligand-binding proteins to create aaRSs whose metabolic labeling activities depend upon post-translational interactions with ligands.
转录控制可用于通过调节正交氨酰-tRNA合成酶（aaRS）的表达来对细胞进行编程，以便用非标准氨基酸标记蛋白质。然而，我们目前还无法对细胞进行编程，使其根据aaRS和配体结合来控制标记。为了鉴定其活性可通过与配体相互作用来调节的aaRS，我们采用了一种组合方法来发现大肠杆菌甲硫氨酰-tRNA合成酶（MetRS）的片段化变体，这些变体需要与结合蛋白融合才能达到最大活性。我们发现，可以利用这些分裂蛋白通过两种方法创建配体依赖性MetRS。当一对MetRS片段与FKBP12和mTOR的FKBP-雷帕霉素结合结构域（FRB）融合，并引入将底物特异性导向叠氮亮氨酸（Anl）的突变时，在含有雷帕霉素的生长培养基中，Anl代谢标记显著增强，雷帕霉素可稳定FKBP12-FRB复合物。此外，将MetRS片段融合到雌激素受体配体结合结构域的末端，得到的蛋白质在向生长培养基中添加4-羟基他莫昔芬（4-HT）时，其Anl代谢标记显著增强。这些发现表明，分裂的MetRS可以与一系列配体结合蛋白融合，以创建其代谢标记活性取决于与配体的翻译后相互作用的aaRS。
