Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes
Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes
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DOI：
10.1007/s10858-006-0008-8
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发表时间：
2006-03-01
影响因子：
2.7
通讯作者：
Shimada, I
中科院分区：
文献类型：
--
作者：
Takahashi, H;Miyazawa, M;Shimada, I
与涉及化学位移扰动和氢-氘交换实验的传统 NMR 方法相比，交叉饱和实验可以更严格地鉴定大型蛋白质复合物（MW > 50 K）的接触残基 [Takahashi 等人，2017]。 (2000) 国家。结构。生物学，7，220-223]。在基于酰胺质子的交叉饱和实验中，结合使用配体蛋白的不可交换质子的高氘化水平和低浓度(H2O)-H-1的溶剂大大增强了分子间交叉饱和现象的选择性。不幸的是，实验的限制导致了灵敏度的损失。此外，由于主链酰胺质子通常不暴露于溶剂，因此针对主链酰胺质子的饱和转移效率不是很高。在这里，我们提出了另一种交叉饱和实验，该实验利用配体蛋白侧链的甲基质子。由于沿着甲基轴的快速内旋转，我们从理论上和实验上证明了这种方法的效率提高。与基于酰胺质子的方法相比，利用甲基的交叉饱和实验在灵敏度和饱和转移效率方面具有明显的优势。
Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW > 50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of (H2O)-H-1 greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach.