Abstract
FoF1-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H+ translocation in membranes. The Fo c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13C NMR (ssNMR) signals of the Fo c-subunit ring of thermophilic Bacillus PS3 (TFo c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional Cα(i+1)–C′Cα(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue 13C–13C chemical shift correlations, 97% of Cα, 97% of Cβ and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TFo c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TFo c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.
Similar content being viewed by others
References
Bak S, Kang SJ, Suzuki T, Yoshida M, Fujiwara T, Akutsu H (2013) Improved purification of thermophilic FoF1-ATP synthase c-subunit rings and solid-state NMR characterization of them in different lipid membranes. J Korean Magn Res Soc 17:67–75
Balakrishna AM, Seelert H, Marx SH, Dencher NA, Gruber G (2014) Crystallographic structure of the turbine c-ring from spinach chloroplast F-ATP synthase. Biosci Rep 34:147–154
Etzkorn M, Martell S, Andronesi OC, Seidel K, Engelhard M, Baldus M (2007) Secondary structure, dynamics, and topology of a seven-helix receptor in native membranes, studied by solid-state NMR spectroscopy. Angew Chem Int Ed 46:459–462
Fritzsching KJ, Yang Y, Schmidt-Rohr K, Hong M (2013) Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information. J Biomol NMR 56:155–167
Fujiwara T, Todokoro Y, Yanagishita H, Tawarayama M, Kohno T, Wakamatsu K, Akutsu H (2004) Signal assignments and chemical-shift structural analysis of uniformly 13C, 15N-labeled peptide, mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning. J Biomol NMR 28:311–325
Goddard TD, Kneller DG (2008) SPARKY 3. University of California San Francisco
Heise H, Hoyer W, Becker S, Andronesi OC, Riedel D, Baldus M (2005) Molecular-level secondary structure, polymorphism, and dynamics of full-length α-synuclein fibrils studied by solid-state NMR. Proc Natl Acad Sci USA 102:15871–15876
Higman ,VA, Flinders J, Hiller M, Jehle S, Markovic S, Fiedler S, van Rossum B-J, Oschkinat H (2009) Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins. J Biomol NMR 44:245–260
Iwai H, Fiaux J (2007) Use of biosynthetic fractional 13C-labeling for backbone NMR assignment of proteins. J Biomol NMR 37:187–193
Kang SJ, Todokoro Y, Yumen I, Shen B, Iwasaki I, Suzuki T, Miyagi A, Yoshida M, Fujiwara T, Akutsu H (2014) Active-site structure of thermophilic Fo c-subunit ring in membranes elucidated by solid-state NMR. Biophys J 106:390–398
Matthies D, Preiss L, Klyszejko AL, Muller DJ, Cook GM, Vonck J, Meier T (2009) The c13 ring from a thermoalkaliphilic ATP synthase reveals an extended diameter due to a special structural region. J Mol Biol 388:611–618
Meier T, Polzer P, Diederichs K, Welte W, Dimroth P (2005) Structure of the rotor ring of F-Type Na+-ATPase from Ilyobacter tartaricus. Science 308:659–662
Mitome N, Suzuki T, Hayashi S, Yoshida M (2004) Thermophilic ATP synthase has a decamer c-ring: indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling. Proc Natl Acad Sci USA 101:12159–12164
Nakano T, Ikegami T, Suzuki T, Yoshida M, Akutsu, H (2006) A new solution structure of ATP synthase subunit c from thermophilic Bacillus PS3, suggesting a local conformational change for H+-translocation. J Mol Biol 358:132–144
Nesci S, Trombetti F, Ventrella V, Pagliarani A (2016) The c-ring of the F1Fo-ATP synthase: facts and perspective. J Membr Biol 249:11–21
Pogoryelov D, Yildiz O, Faraldo-Gomez JD, Meier T (2009) High-resolution structure of the rotor ring of a proton-dependent ATP synthase. Nat Struct Mol Biol 16:1068–1073
Preiss L, Yildiz O, Hicks DB, Krulwich TA, Meier T (2010) A new type of proton coordination in an F1Fo-ATP synthase rotor ring. PLoS Biol 8:e1000443
Preiss L, Langer JD, Yildiz Ö, Eckhardt-Strelau L, .Guillemont JEG, Koul A, Meier T (2015) Structure fo the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci Adv 1:e1500106, 1–8
Rastogi VK, Girvin ME (1999) Structural changes linked to proton translocation by subunit c of the ATP synthase. Nature 402:263–268
Shen Y, Bax A (2010) Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts. J Biomol NMR 46:199–204
Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J Biomol NMR 56:227–241. https://spin.niddk.nih.gov/bax/software/TALOS-N/
Stock D, Leslie AG, Walker JE (1999) Molecular architecture of the rotary motor in ATP synthase. Science 286:1700–1705
Symersky J, Pagadala V, Osowski D, Krah A, Meier T, Faraldo-Gomez JD, Mueller DM (2012) Structure of the c10 ring of the yeast mitochondrial ATP synthase in the open conformation. Nat Struct Mol Biol 19:485–491, S481
Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637
Vollmar M, Schlieper D, Winn M, Buchner C, Groth G (2009) Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase. J Biol Chem 284:18228–18235
Watt IN, Montgomery MG, Runswick MJ, Leslie AG, Walker JE (2010) Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc Natl Acad Sci USA 107:16823–16827
Waugh DS (1996) Genetic tools for selective labeling of proteins with α-15N-amino acids. J Biomol NMR 8:184–192
Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical shift data. J Biomol NMR 4:171–180
Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes (1995) B.D. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81
Wuister GW, Kim SJ, Wu C, Bax A (1994) 2D and 3D NMR study of phenylalanine residues in proteins by reverse isotope labeling. J Am Chem Soc 116:9206–9210
Yoshida M, Muneyuki E, Hisabori T (2001) ATP synthase—a marvellous rotary engine of the cell. Nat Rev Mol Cell Biol 2:669–677
Yumen I, Iwasaki I, Suzuki T, Todokoro Y, Tanaka K, Okada O, Fujiwara T, Yoshida M, Akutsu H (2012) Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli. Protein Expr Purif 82:396–401
Acknowledgements
We would like to thank Ms. Ikuko Yumen and Iku Iwasaki for their assistance in sample preparation. We are also grateful to Profs. Se-Won Suh, Bong-Jin Lee (Seoul National University), and Takahisa Ikegami (Yokohama City University) for their encouragement and support. This work was partly supported by the Targeted Proteins Research Program (HA, TF, and MY) from Ministry of Education, Culture, Sports, Science, and Technology, Japan, and a World Class University Grant from the Korean Research Foundation. SJK has been a member of an international collaborative research project of the Institute for Protein Research, Osaka University from 2011 to 2013.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kang, SJ., Todokoro, Y., Bak, S. et al. Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure. J Biomol NMR 70, 53–65 (2018). https://doi.org/10.1007/s10858-017-0158-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10858-017-0158-x