Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan; Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehirocho, Tsurumi-ku, Yokohama 230-0045, Japan.
Biophys Chem. 2023 May;296:106988. doi: 10.1016/j.bpc.2023.106988. Epub 2023 Mar 1.
Nuclear magnetic resonance (NMR) investigation of large membrane proteins requires well-focused questions and critical techniques. Here, research strategies for FF-ATP synthase, a membrane-embedded molecular motor, are reviewed, focusing on the β-subunit of F-ATPase and c-subunit ring of the enzyme. Segmental isotope-labeling provided 89% assignment of the main chain NMR signals of thermophilic Bacillus (T)Fβ-monomer. Upon nucleotide binding to Lys164, Asp252 was shown to switch its hydrogen-bonding partner from Lys164 to Thr165, inducing an open-to-closed bend motion of TFβ-subunit. This drives the rotational catalysis. The c-ring structure determined by solid-state NMR showed that cGlu56 and cAsn23 of the active site took a hydrogen-bonded closed conformation in membranes. In 505 kDa TFF, the specifically isotope-labeled cGlu56 and cAsn23 provided well-resolved NMR signals, which revealed that 87% of the residue pairs took a deprotonated open conformation at the Fa-c subunit interface, whereas they were in the closed conformation in the lipid-enclosed region.
核磁共振（NMR）研究大型膜蛋白需要有针对性的问题和关键技术。本文综述了 FF-ATP 合酶（一种膜嵌入的分子马达）的研究策略，重点介绍了 F-ATP 酶的β亚基和酶的 c 亚基环。分段同位素标记提供了嗜热芽孢杆菌（T）Fβ-单体的主链 NMR 信号的 89%的分配。在核苷酸结合到 Lys164 后，Asp252 被证明将其氢键供体从 Lys164 切换到 Thr165，诱导 TFβ-亚基的开-闭弯曲运动。这驱动了旋转催化。通过固态 NMR 确定的 c 环结构表明，活性位点的 cGlu56 和 cAsn23 在膜中采取氢键封闭构象。在 505 kDa TFF 中，特异性同位素标记的 cGlu56 和 cAsn23 提供了分辨率良好的 NMR 信号，揭示了 Fa-c 亚基界面上 87%的残基对采取去质子化的开放构象，而在脂质封闭区域中它们处于封闭构象。
