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Active-site structure of the thermophilic Foc-subunit ring in membranes elucidated by solid-state NMR.

ABSTRACT: 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 thermophilic c-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. TFoc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TFoc 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 (13)C-(13)C correlation spectra of TFoc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C(?)i+1-C(?)i correlation spectrum of specifically (13)C,(15)N-labeled TFoc rings. The C(?) 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(?) 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 TFoc ring, suggesting two different means of proton storage in the c rings.

SUBMITTER: Kang SJ

PROVIDER: S-EPMC3907233 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Active-site structure of the thermophilic Foc-subunit ring in membranes elucidated by solid-state NMR.

Kang Su-Jin SJ Todokoro Yasuto Y Yumen Ikuko I Shen Bo B Iwasaki Iku I Suzuki Toshiharu T Miyagi Atsushi A Yoshida Masasuke M Fujiwara Toshimichi T Akutsu Hideo H

Biophysical journal 20140101 2

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 thermophilic c-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. TFoc oligomers were purified using a virtual ring His tag. The membran ...[more]

PMID: 24461014

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Project description:Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys(40), residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein dynamics but also protein-lipid interactions in detail.

Dataset Information

Active-site structure of the thermophilic Foc-subunit ring in membranes elucidated by solid-state NMR.

Publications

Active-site structure of the thermophilic Foc-subunit ring in membranes elucidated by solid-state NMR.

Similar Datasets

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