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Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids
Journal of Medicinal Chemistry ( IF 6.8 ) Pub Date : 2023-03-15 , DOI: 10.1021/acs.jmedchem.3c00222
Kasuni B Ekanayake 1 , Mithun C Mahawaththa 1 , Haocheng Qianzhu 2 , Elwy H Abdelkader 1 , Josemon George 2 , Sven Ullrich 2 , Rhys B Murphy 2 , Sarah E Fry 3, 4 , Jason Johansen-Leete 3, 4 , Richard J Payne 3, 4 , Christoph Nitsche 2 , Thomas Huber 2 , Gottfried Otting 1
Journal of Medicinal Chemistry ( IF 6.8 ) Pub Date : 2023-03-15 , DOI: 10.1021/acs.jmedchem.3c00222
Kasuni B Ekanayake 1 , Mithun C Mahawaththa 1 , Haocheng Qianzhu 2 , Elwy H Abdelkader 1 , Josemon George 2 , Sven Ullrich 2 , Rhys B Murphy 2 , Sarah E Fry 3, 4 , Jason Johansen-Leete 3, 4 , Richard J Payne 3, 4 , Christoph Nitsche 2 , Thomas Huber 2 , Gottfried Otting 1
Affiliation
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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.
中文翻译:
通过基因编码的非规范氨基酸的核磁共振波谱探测大蛋白质上的配体结合位点
N 6 -(((三甲基甲硅烷基)-甲氧基)羰基) -l-赖氨酸(TMSK)和N 6 -三氟乙酰基-l-赖氨酸(TFAK)是非规范氨基酸,可以通过基因编码安装在蛋白质中。此外,我们描述了一种特异于N 6 -(((三甲基甲硅烷基)甲基)-氨基甲酰基) -l-赖氨酸 (TMSNK) 的新型氨酰基-tRNA 合成酶,其化学性质比 TMSK 更稳定。使用二聚体 SARS-CoV-2 主蛋白酶 (M pro ) 作为具有三种不同配体的模型系统,我们发现溶剂暴露的三甲基甲硅烷基和 CF 3基团的1 H 和19 F 核在核磁中产生强烈信号共振(NMR)谱。当它们位于活性位点附近而不是远离活性位点时,它们对活性位点配体的反应显着不同。相反,NMR 探针未能证实先前报道的配体 pelitinib 的结合位点,该配体被发现可通过促进酶活性二聚体的形成来增强 M pro的活性。总之,氨基酸 TMSK、TMSNK 和 TFAK 为配体在低浓度下与稳定性有限的大蛋白结合的位点特异性 NMR 分析开辟了一条有吸引力的途径。
更新日期:2023-03-15
中文翻译:
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通过基因编码的非规范氨基酸的核磁共振波谱探测大蛋白质上的配体结合位点
N 6 -(((三甲基甲硅烷基)-甲氧基)羰基) -l-赖氨酸(TMSK)和N 6 -三氟乙酰基-l-赖氨酸(TFAK)是非规范氨基酸,可以通过基因编码安装在蛋白质中。此外,我们描述了一种特异于N 6 -(((三甲基甲硅烷基)甲基)-氨基甲酰基) -l-赖氨酸 (TMSNK) 的新型氨酰基-tRNA 合成酶,其化学性质比 TMSK 更稳定。使用二聚体 SARS-CoV-2 主蛋白酶 (M pro ) 作为具有三种不同配体的模型系统,我们发现溶剂暴露的三甲基甲硅烷基和 CF 3基团的1 H 和19 F 核在核磁中产生强烈信号共振(NMR)谱。当它们位于活性位点附近而不是远离活性位点时,它们对活性位点配体的反应显着不同。相反,NMR 探针未能证实先前报道的配体 pelitinib 的结合位点,该配体被发现可通过促进酶活性二聚体的形成来增强 M pro的活性。总之,氨基酸 TMSK、TMSNK 和 TFAK 为配体在低浓度下与稳定性有限的大蛋白结合的位点特异性 NMR 分析开辟了一条有吸引力的途径。