Advertisement

ATP Synthase: Expression, Purification, and Function

  • Meghna Sobti
  • Robert Ishmukhametov
  • Alastair G. StewartEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2073)

Abstract

ATP synthase is an essential enzyme found in all known forms of life, generating the majority of cellular energy via a rotary catalytic mechanism. Here, we describe the in-depth methods for expression, purification, and functional assessment of E. coli ATP synthase.

Key words

ATPase ATP synthase Rotary motor Enzyme Bioenergetics 

References

  1. 1.
    Stewart AG, Laming EM, Sobti M, Stock D (2014) Rotary ATPases–dynamic molecular machines. Curr Opin Struct Biol 25:40–48.  https://doi.org/10.1016/j.sbi.2013.11.013CrossRefPubMedGoogle Scholar
  2. 2.
    Stewart AG (2014) The molecular V brake. J Mol Biol 426:273–274.  https://doi.org/10.1016/j.jmb.2013.10.003CrossRefPubMedGoogle Scholar
  3. 3.
    Stewart AG, Stock D (2012) Priming a molecular motor for disassembly. Structure 20:1799–1800.  https://doi.org/10.1016/j.str.2012.10.003CrossRefPubMedGoogle Scholar
  4. 4.
    Stewart AG, Sobti M, Harvey RP, Stock D (2013) Rotary ATPases: models, machine elements and technical specifications. BioArchitecture 3:2–12.  https://doi.org/10.4161/bioa.23301CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Sielaff H, Duncan TM, Borsch M (2018) The regulatory subunit epsilon in Escherichia coli FOF1-ATP synthase. Biochim Biophys Acta Bioenerg 1859:775–788.  https://doi.org/10.1016/j.bbabio.2018.06.013CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Deckers-Hebestreit G, Greie J, Stalz W, Altendorf K (2000) The ATP synthase of Escherichia coli: structure and function of F0 subunits. Biochim Biophys Acta 1458:364–373CrossRefGoogle Scholar
  7. 7.
    Wilkens S, Capaldi RA (1998) Electron microscopic evidence of two stalks linking the F1 and F0 parts of the Escherichia coli ATP synthase. Biochim Biophys Acta 1365:93–97CrossRefGoogle Scholar
  8. 8.
    Boyer PD (1997) The ATP synthase–a splendid molecular machine. Annu Rev Biochem 66:717–749.  https://doi.org/10.1146/annurev.biochem.66.1.717CrossRefPubMedGoogle Scholar
  9. 9.
    Capaldi RA, Schulenberg B, Murray J, Aggeler R (2000) Cross-linking and electron microscopy studies of the structure and functioning of the Escherichia coli ATP synthase. J Exp Biol 203:29–33PubMedGoogle Scholar
  10. 10.
    Jiang W, Fillingame RH (1998) Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking. Proc Natl Acad Sci U S A 95:6607–6612CrossRefGoogle Scholar
  11. 11.
    Lightowlers RN, Howitt SM, Hatch L, Gibson F, Cox GB (1987) The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit. Biochim Biophys Acta 894:399–406CrossRefGoogle Scholar
  12. 12.
    Cingolani G, Duncan TM (2011) Structure of the ATP synthase catalytic complex F1 from Escherichia coli in an autoinhibited conformation. Nat Struct Mol Biol 18:701–707.  https://doi.org/10.1038/nsmb.2058CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sobti M et al (2016) Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states. Elife 5.  https://doi.org/10.7554/eLife.21598
  14. 14.
    Sobti M et al (2019) Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP. Elife 8.  https://doi.org/10.7554/eLife.43864
  15. 15.
    Klionsky DJ, Brusilow WS, Simoni RD (1984) In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli. J Bacteriol 160:1055–1060PubMedPubMedCentralGoogle Scholar
  16. 16.
    Ishmukhametov R, Galkin MA, Vik SB (2005) Ultrafast purification and reconstitution of his-tagged cysteine-less Escherichia coli F1Fo ATP synthase. Biochim Biophys Acta 1706:110–116.  https://doi.org/10.1016/j.bbabio.2004.09.012CrossRefPubMedGoogle Scholar
  17. 17.
    Rubinstein JL (2007) Structural analysis of membrane protein complexes by single particle electron microscopy. Methods 41:409–416.  https://doi.org/10.1016/j.ymeth.2006.07.019CrossRefPubMedGoogle Scholar
  18. 18.
    Warren GB, Toon PA, Birdsall NJ, Lee AG, Metcalfe JC (1974) Reconstitution of a calcium pump using defined membrane components. Proc Natl Acad Sci U S A 71:622–626CrossRefGoogle Scholar
  19. 19.
    Lotscher HR, deJong C, Capaldi RA (1984) Interconversion of high and low adenosinetriphosphatase activity forms of Escherichia coli F1 by the detergent lauryldimethylamine oxide. Biochemistry 23:4140–4143CrossRefGoogle Scholar
  20. 20.
    Linnett PE, Beechey RB (1979) Inhibitors of the ATP synthethase system. Methods Enzymol 55:472–518CrossRefGoogle Scholar
  21. 21.
    Ishmukhametov RR, Russell AN, Berry RM (2016) A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes. Nat Commun 7:13025.  https://doi.org/10.1038/ncomms13025CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Meghna Sobti
    • 1
  • Robert Ishmukhametov
    • 2
  • Alastair G. Stewart
    • 1
    Email author
  1. 1.The Victor Chang Cardiac Research InstituteDarlinghurstAustralia
  2. 2.University of OxfordOxfordUK

Personalised recommendations