Genetic Encoding of N6-(((Trimethylsilyl)methoxy)carbonyl)-L-lysine for NMR Studies of Protein-Protein and Protein-Ligand Interactions
Genetic Encoding of N6-(((Trimethylsilyl)methoxy)carbonyl)-L-lysine for NMR Studies of Protein-Protein and Protein-Ligand Interactions
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DOI：
10.1021/jacs.0c11971
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发表时间：
2021-01-05
影响因子：
15
通讯作者：
Otting, Gottfried
中科院分区：
文献类型：
--
作者：
Abdelkader, Elwy H.;Qianzhu, Haocheng;Otting, Gottfried
三甲基甲硅烷基（TMS）基团是生物大分子的优秀NMR探针，因为它们在0 ppm附近的H-1 NMR光谱中产生强烈的单线态，而几乎没有其他质子共振发生。我们报告了一个用于位点特异性掺入蛋白质的N-6-（三甲基甲硅烷基）-甲氧基）羰基）-l-赖氨酸（TMSK）的遗传编码系统。该系统基于吡咯赖氨酰-tRNA合成酶突变体，其在体内和无细胞蛋白质合成中以高产率和纯度递送蛋白质。由于TMS信号可以很容易地在高分子量体系的1D H-1 NMR谱中识别，而不需要同位素标记，TMSK为蛋白质结构和功能的研究提供了一个很好的位点特异性NMR探针，这既便宜又方便。我们证明了实用的TMSK检测配体结合，测量构象变化的速率，并评估蛋白质二聚顺磁弛豫增强。此外，我们提出了一个系统，用于在相同的蛋白质中的两个不同的非天然氨基酸（TMSK和O-叔丁基酪氨酸）的双掺入量足够的NMR光谱。TMS和叔丁基基团的紧密接近很容易通过核Overhauser效应检测到。
Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in H-1 NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N-6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D H-1 NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.