The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 949–956 | Cite as

Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals

  • Wanatchaporn Arunmanee
  • J. Robin Harris
  • Jeremy H. LakeyEmail author


Amphipols (APol) are polymers which can solubilise and stabilise membrane proteins (MP) in aqueous solutions. In contrast to conventional detergents, APol are able to keep MP soluble even when the free APol concentration is very low. Outer membrane protein F (OmpF) is the most abundant MP commonly found in the outer membrane (OM) of Escherichia coli. It plays a vital role in the transport of hydrophilic nutrients, as well as antibiotics, across the OM. In the present study, APol was used to solubilise OmpF to characterize its interactions with molecules such as lipopolysaccharides (LPS) or colicins. OmpF was reconstituted into APol by the removal of detergents using Bio-Beads followed by size-exclusion chromatography (SEC) to remove excess APol. OmpF/APol complexes were then analysed by SEC, dynamic light scattering (DLS) and transmission electron microscopy (TEM). TEM showed that in the absence of free APol–OmpF associated as long filaments with a thickness of ~6 nm. This indicates that the OmpF trimers lie on their sides on the carbon EM grid and that they also favour side by side association. The formation of filaments requires APol and occurs very rapidly. Addition of LPS to OmpF/APol complexes impeded filament formation and the trimers form 2D sheets which mimic the OM. Consequently, free APol is undoubtedly required to maintain the homogeneity of OmpF in solutions, but ‘minimum APol’ provides a new phase, which can allow weaker protein–protein and protein–lipid interactions characteristic of native membranes to take place and thus control 1D–2D crystallisation.


OmpF Lipopolysaccharides Amphipol Transmission electron microscopy Dynamic light scattering SEC 



This work was supported by a Royal Thai Government Scholarship to WA and the Wellcome Trust (Grant number 093581). We thank the Newcastle University Biomedical Electron Microscopy Unit and Dr. Helen Ridley for her technical assistance. We thank Jean-Luc Popot and Christophe Tribet for advice and amphipol samples.


  1. Althoff T, Mills DJ, Popot J-L, Kuehlbrandt W (2011) Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30(22):4652–4664CrossRefGoogle Scholar
  2. Baboolal TG, Conroy MJ, Gill K, Ridley H, Visudtiphole V, Bullough PA, Lakey JH (2008) Colicin N binds to the periphery of its receptor and translocator, outer membrane protein F. Structure 16(3):371–379CrossRefGoogle Scholar
  3. Cao E, Liao M, Cheng Y, Julius D (2013) TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504(7478):113CrossRefGoogle Scholar
  4. Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D (2007) Colicin biology. Microbiol Mol Biol Rev 71(1):158–229CrossRefGoogle Scholar
  5. Catoire LJ, Zoonens M, Van Heijenoort C, Giusti F, Guittet É, Popot JL (2010) Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX. Eur Biophys J 39(4):623–630CrossRefGoogle Scholar
  6. Cisneros DA, Muller DJ, Daud SM, Lakey JH (2006) An approach to prepare membrane proteins for single-molecule imaging. Angew Chem Int Ed Engl 45(20):3252–3256CrossRefGoogle Scholar
  7. Clifton LA, Johnson CL, Solovyova AS, Callow P, Weiss KL, Ridley H, Le Brun AP, Kinane CJ, Webster JRP, Holt SA, Lakey JH (2012) Low resolution structure and dynamics of a colicin-receptor complex determined by neutron scattering. J Biol Chem 287(1):337–346CrossRefGoogle Scholar
  8. Cowan SW, Schirmer T, Rummel G, Steiert M, Ghosh R, Paupit RA, Jansonius JN, Rosenbusch JP (1992) Crystal structures explain functional properties of two E. coli porins. Nature 358:727–733CrossRefGoogle Scholar
  9. Cvetkov TL, Huynh KW, Cohen MR, Moiseenkova-Bell VY (2011) Molecular architecture and subunit organization of TRPA1 ion channel revealed by electron microscopy. J Biol Chem 286(44):38168–38176CrossRefGoogle Scholar
  10. Evans LJA, Cooper A, Lakey JH (1996) Direct measurement of the association of a protein with a family of membrane receptors. J Mol Biol 255(4):559–563CrossRefGoogle Scholar
  11. Flötenmeyer M, Weiss H, Tribet C, Popot JL, Leonard K (2007) The use of amphipathic polymers for cryo electron microscopy of NADH:ubiquinone oxidoreductase (complex I). J Microsc 227(3):229–235CrossRefGoogle Scholar
  12. Gohon Y, Dahmane T, Ruigrok RWH, Schuck P, Charvolin D, Rappaport F, Timmins P, Engelman DM, Tribet C, Popot J-L, Ebel C (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J 94(9):3523–3537CrossRefGoogle Scholar
  13. Harris JR (1997) Negative staining and cryoelectron microscopy. Bios Scientific Publishers Ltd., OxfordCrossRefGoogle Scholar
  14. Hoenger A, Ghosh R, Schoenenberger CA, Aebi U, Engel A (1993a) Direct in-situ structural-analysis of recombinant outer-membrane porins expressed in an OmpA-deficient mutant Escherichia coli strain. J Struct Biol 111(3):212–221CrossRefGoogle Scholar
  15. Hoenger A, Pagès J-M, Fourel D, Engel A (1993b) The orientation of porin OmpF in the outer membrane of Escherichia coli. J Mol Biol 233(3):400–413CrossRefGoogle Scholar
  16. Jaroslawski S, Duquesne K, Sturgis JN, Scheuring S (2009) High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans. Mol Microbiol 74(5):1211–1222CrossRefGoogle Scholar
  17. Johnson CL, Ridley H, Pengelly RJ, Salleh MZ, Lakey JH (2013) The unstructured domain of colicin N kills Escherichia coli. Mol Microbiol 89(1):84–95CrossRefGoogle Scholar
  18. Lakey JH, Watts JP, Lea EJA (1985) Characterization of channels induced in planar bilayer-membranes by detergent solubilized Escherichia coli porins. Biochim Biophys Acta 817(2):208–216CrossRefGoogle Scholar
  19. Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504(7478):107CrossRefGoogle Scholar
  20. Nakae T (1976) Outer membrane of Salmonella. Isolation of protein complex that produces transmembrane channels. J Biol Chem 251(7):2176–2178PubMedGoogle Scholar
  21. Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67(4):593CrossRefGoogle Scholar
  22. Popot J-L (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions. In: Kornberg RD, Raetz CRH, Rothman JE, Thorner JW (eds) Annual review of biochemistry, vol 79., pp 737–775Google Scholar
  23. Popot JL, Berry EA, Charvolin D, Creuzenet C, Ebel C, Engelman DM, Flotenmeyer M, Giusti F, Gohon Y, Herve P, Hong Q, Lakey JH, Leonard K, Shuman HA, Timmins P, Warschawski DE, Zito F, Zoonens M, Pucci B, Tribet C (2003) Amphipols: polymeric surfactants for membrane biology research. Cell Mol Life Sci 60(8):1559–1574CrossRefGoogle Scholar
  24. Rosenbusch JP (1974) Characterisation of the major envelope protein fron Escherichia coli. J Biol Chem 249:8019–8029PubMedGoogle Scholar
  25. Schabert FA, Engel A (1994) Reproducible acquisition of Escherichia coli porin surface topographs by atomic-force microscopy. Biophys J 67(6):2394–2403CrossRefGoogle Scholar
  26. Schneck E, Schubert T, Konovalov OV, Quinn BE, Gutsmann T, Brandenburg K, Oliveira RG, Pink DA, Tanaka M (2010) Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence. Proc Natl Acad Sci USA 107(20):9147–9151CrossRefGoogle Scholar
  27. Tribet C, Audebert R, Popot J-L (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93(26):15047–15050CrossRefGoogle Scholar
  28. Tribet C, Audebert R, Popot JL (1997) Stabilization of hydrophobic colloidal dispersions in water with amphiphilic polymers: application to integral membrane proteins. Langmuir 13(21):5570–5576CrossRefGoogle Scholar
  29. Tribet C, Mills D, Haider M, Popot JL (1998) Scanning transmission electron microscopy study of the molecular mass of amphipol cytochrome b(6)f complexes. Biochimie 80(5–6):475–482CrossRefGoogle Scholar
  30. Visudtiphole V, Thomas MB, Chalton DA, Lakey JH (2005) Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers. Biochem J 392:375–381CrossRefGoogle Scholar
  31. White SH (2009) Biophysical dissection of membrane proteins. Nature 459(7245):344–346CrossRefGoogle Scholar
  32. Zoonens M, Catoire LJ, Giusti F, Popot JL (2005) NMR study of a membrane protein in detergent-free aqueous solution. Proc Natl Acad Sci USA 102(25):8893–8898CrossRefGoogle Scholar
  33. Zoonens M, Giusti F, Zito F, Popot J-L (2007) Dynamics of membrane protein/amphipol association studied by forster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46(36):10392–10404CrossRefGoogle Scholar

Copyright information

© The Author(s) 2014

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Wanatchaporn Arunmanee
    • 1
  • J. Robin Harris
    • 1
    • 2
  • Jeremy H. Lakey
    • 1
    Email author
  1. 1.Institute for Cell and Molecular BiosciencesNewcastle UniversityNewcastle upon TyneUK
  2. 2.Institute of ZoologyUniversity of MainzMainzGermany

Personalised recommendations