Active-site structure of the thermophilic foc-subunit ring in membranes elucidated by solid-state NMR.
Biophys J. 2014 Jan 21;106(2):390-8
Authors: Kang SJ, Todokoro Y, Yumen I, Shen B, Iwasaki I, Suzuki T, Miyagi A, Yoshida M, Fujiwara T, Akutsu H
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.
Irregular structure of the HIV fusion peptide in membranes demonstrated by solid-state NMR and MD simulations.
Irregular structure of the HIV fusion peptide in membranes demonstrated by solid-state NMR and MD simulations.
Irregular structure of the HIV fusion peptide in membranes demonstrated by solid-state NMR and MD simulations.
Eur Biophys J. 2011 Jan 28;
Authors: Grasnick D, Sternberg U, Strandberg E, Wadhwani P, Ulrich AS
To better understand peptide-induced membrane fusion at a molecular level, we set out to determine the structure of the fusogenic peptide FP23 from the HIV-1 protein gp41 when bound to a lipid bilayer. An established solid-state...
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01-29-2011 12:35 PM
Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR.
Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR.
Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR.
J Biomol NMR. 2010 Sep;48(1):1-11
Authors: Todokoro Y, Kobayashi M, Sato T, Kawakami T, Yumen I, Aimoto S, Fujiwara T, Akutsu H
The subunit c-ring of H(+)-ATP synthase (F(o) c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we...
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12-18-2010 12:00 PM
[NMR paper] Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase reveal
Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy.
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J Am Chem Soc. 2005 Jun 15;127(23):8424-32
Authors: Wu J, Bell AF, Jaye AA, Tonge PJ
Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig...
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[NMR paper] Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state
Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant.
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Biophys J. 2002 Feb;82(2):1017-29
Authors: Hatcher ME, Hu JG, Belenky M, Verdegem P, Lugtenburg J, Griffin RG, Herzfeld J
By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of...
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11-24-2010 08:49 PM
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Biochemistry. 2000 Jun 13;39(23):6751-5
Authors: Matysik J, Alia A, Nachtegaal G, van Gorkom HJ, Hoff AJ, de Groot HJ
Calcium (Ca(2+)) is an essential cofactor for photosynthetic oxygen evolution. Although the involvement of Ca(2+) at the oxidizing side of photosystem II of plants has been known for a long time, its ligand interactions and mode of...
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J Biol Chem. 1991 Aug 15;266(23):15001-8
Authors: de Ropp JS, La Mar GN, Wariishi H, Gold MH
One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting...
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[NMR paper] NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase
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J Biol Chem. 1991 Aug 15;266(23):15001-8
Authors: de Ropp JS, La Mar GN, Wariishi H, Gold MH
One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting...
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08-21-2010 11:12 PM
Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthas
Abstract The subunit c-ring of H+-ATP synthase (Fo c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly -labeled Fo c from E. coli (EFo c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the 13C and 15N signals were assigned. The obtained chemical shifts suggested that EFo c takes on a hairpin-type helix-loop-helix...
Active-site structure of the thermophilic foc-subunit ring in membranes elucidated by solid-state NMR.
Linear Mode
