Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids

N6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and N6-trifluoroacetyl-l-lysine (TFAK) are non-canonical amino acids, which can be installed in proteins by genetic encoding. In addition, we describe a new aminoacyl-tRNA synthetase specific for N6-(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK), which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2 main protease (Mpro) as a model system with three different ligands, we show that the 1H and 19F nuclei of the solvent-exposed trimethylsilyl and CF3 groups produce intense signals in the nuclear magnetic resonance (NMR) spectrum. Their response to active-site ligands differed significantly when positioned near rather than far from the active site. Conversely, the NMR probes failed to confirm the previously reported binding site of the ligand pelitinib, which was found to enhance the activity of Mpro by promoting the formation of the enzymatically active dimer. In summary, the amino acids TMSK, TMSNK, and TFAK open an attractive path for site-specific NMR analysis of ligand binding to large proteins of limited stability and at low concentrations.N6-(((三甲基硅基)甲氧基)羰基)-L-赖氨酸（N6-(((trimethylsilyl)-methoxy)carbonyl)-L-lysine, TMSK）与N6-三氟乙酰基-L-赖氨酸（N6-trifluoroacetyl-L-lysine, TFAK）均为非经典氨基酸（non-canonical amino acids），可通过遗传编码方式引入蛋白质中。此外，本文报道了一种针对N6-(((三甲基硅基)甲基)氨基甲酰基)-L-赖氨酸（N6-(((trimethylsilyl)methyl)-carbamoyl)-L-lysine, TMSNK）的特异性新型氨酰-tRNA合成酶（aminoacyl-tRNA synthetase），该底物的化学稳定性优于TMSK。以二聚体严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）主蛋白酶（main protease, Mpro）为模型体系，搭配三种不同配体，研究发现溶剂暴露的三甲基硅基与三氟甲基的1H和19F原子核在核磁共振（nuclear magnetic resonance, NMR）谱中可产生强信号。当这些基团位于活性位点附近而非远端时，其对活性位点配体的响应存在显著差异。反之，该NMR探针未能验证此前报道的吡替尼（pelitinib）结合位点——研究发现吡替尼可通过促进酶活性二聚体的形成，增强Mpro的活性。综上，TMSK、TMSNK与TFAK这三种氨基酸为针对稳定性有限且低浓度大型蛋白质的配体结合位点特异性NMR分析提供了极具潜力的途径。