Active-Site Structure of Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR
Active-Site Structure of Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR
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固态核磁共振阐明膜中嗜热焦点亚基环的活性位点结构
DOI：
10.1016/j.bpj.2013.12.005
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发表时间：
2014
期刊：
影响因子：
3.4
通讯作者：
Akutsu H
中科院分区：
文献类型：
--
作者：
Kang S;Todokoro Y;Yumen I;Shen B;Iwasaki I;Suzuki T;Miyagi A;Morikawa K;Yoshida M;Fujiwara T;Akutsu H
FoF 1-ATP合酶利用跨膜的电化学电势或ATP水解来旋转Foc亚基环。为了阐明潜在的机制，我们进行了结构分析，重点是在膜中的嗜热亚基（TFoc）环的活性位点与固态NMR方法开发用于此目的。我们使用立体阵列同位素标记（SAIL）与无细胞系统突出的目标。使用虚拟环His标签纯化TFocoligomer。根据H+-易位活性和高速原子力显微镜图像，证实膜重构的TFocoligomer是与大肠杆菌中表达的TFocoligomer不可区分的环。为了分析活性位点，记录了用SAIL-Glu和-Asn标记的TFocrings的2D 13 C-13 C相关光谱。利用~（13）C，~（15）N标记的TFocrings的Cαi+1-Cα i相关谱，可以进行完整的信号归属。Glu-56的Cδ化学位移和相关的交叉分析表明，Glu-56的羧基在膜上被质子化，与Asn-23形成H+锁定构象。与Asn-23参与T-Focring相比，E. coli cring的Asp-61 Cγ的化学位移表明水分子参与H+锁定，表明T-Focring中有两种不同的质子储存方式。
FoF1-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the Foc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilicc-subunit (TFoc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TFocoligomers were purified using a virtual ring His tag. The membrane-reconstituted TFocoligomer was confirmed to be a ring indistinguishable from that expressed inE. colion the basis of the H+-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D13C-13C correlation spectra of TFocrings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the Cαi+1-Cαicorrelation spectrum of specifically13C,15N-labeled TFocrings. The Cδchemical shift of Glu-56, which is essential for H+translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H+-locked conformation with Asn-23. The chemical shift of Asp-61 Cγof theE. coli cring indicated an involvement of a water molecule in the H+locking, in contrast to the involvement of Asn-23 in the TFocring, suggesting two different means of proton storage in thecrings.