Active-Site Structure of the Thermophilic F^sub o^c-Subunit Ring in Membranes Elucidated by Solid-State NMR

F^sub o^F^sub 1^-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F^sub o^c-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF^sub o^c) ring in membrane...

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Bibliographic Details
Published in:Biophysical journal 2014-01, Vol.106 (2), p.390
Main Authors: Kang, Su-Jin, Todokoro, Yasuto, Yumen, Ikuko, Shen, Bo, Iwasaki, Iku, Suzuki, Toshiharu, Miyagi, Atsushi, Yoshida, Masasuke, Fujiwara, Toshimichi, Akutsu, Hideo
Format: Article
Language:English
Subjects:
Correlation analysis
Membranes
NMR
Nuclear magnetic resonance
Spectrum analysis
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Summary:F^sub o^F^sub 1^-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F^sub o^c-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF^sub o^c) 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. TF^sub o^c oligomers were purified using a virtual ring His tag. The membrane-reconstituted TF^sub o^c oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H+-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D ^sup 13^C-^sup 13^C correlation spectra of TF^sub o^c rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C^sup α^^sub i+1^-C^sup α^^sub i^ correlation spectrum of specifically ^sup 13^C,^sup 15^N-labeled TF^sub o^c rings. The C^sup δ^ chemical 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 C^sup γ^ of the E. coli c ring indicated an involvement of a water molecule in the H+ locking, in contrast to the involvement of Asn-23 in the TF^sub o^c ring, suggesting two different means of proton storage in the c rings. [PUBLICATION ABSTRACT]
ISSN:0006-3495
1542-0086