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The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 1005–1018 | Cite as

Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli: A Biochemical and Structural Case Study

  • Aleksandr Sverzhinsky
  • Shuo Qian
  • Lin Yang
  • Marc Allaire
  • Isabel Moraes
  • Dewang Ma
  • Jacqueline W. Chung
  • Manuela Zoonens
  • Jean-Luc Popot
  • James W. CoultonEmail author
Article

Abstract

Nutrient import across Gram-negative bacteria’s outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB–ExbD–TonB. Having purified the ExbB4–ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4–ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB4–ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB4–ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6–ExbD4, and propose a structural arrangement of its transmembrane α-helical domains.

Keywords

Membrane protein complex Amphipol Detergent EM SAXS/SANS 

Notes

Acknowledgments

Particular thanks are due to F. Giusti (UMR 7099, Paris) for synthesizing the deuterated and the fluorescent amphipols used in this project. This work was supported by an operating grant to J.W.C. from the Canadian Institutes of Health Research (CIHR reference number 200709MOP-178048-BMA-CFAA-11449). The Groupe d’étude des protéines membranaires (GÉPROM), supported by the Fonds de la recherche en santé du Québec (FRSQ), awarded a Projet Novateur to J.W.C. A.S. was awarded fellowships from the CREATE program, Cellular Dynamics of Macromolecular Complexes, Natural Sciences and Engineering Research Council (NSERC) of Canada; from GÉPROM; and from the F.C. Harrison and the Rozanis Funds, Department of Microbiology and Immunology, McGill University. Work in UMR 7099 was supported by the French Centre National de la Recherche Scientifique (CNRS), by Université Paris-7 Denis Diderot, and by grant “DYNAMO”, ANR-11-LABX-0011-01, from the French “Initiative d’Excellence” program. Canada Foundation for Innovation provided infrastructure for the Facility for Electron Microscope Research, McGill University; www.medicine.mcgill.ca/femr/home.html. We appreciate support from Isabelle Rouiller for EM studies. Tara Sprules, manager of the Quebec/Eastern Canada High Field NMR Facility,www.nmrlab.mcgill.ca, guided NMR experiments to quantitate detergent. Research at the Bio-SANS (Center for Structural Molecular Biology) was supported by the U.S. Department of Energy’s Office of Biological and Environmental Research. Research at Oak Ridge National Laboratory’s High Flux Isotope Reactor was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. We appreciate the access to AF4 equipment in the laboratory of Françoise Winnik at the Université de Montréal. This work was facilitated by computing resources from CLUMEQ, under Compute/Calcul Canada. We appreciate laboratory support from Nathalie Croteau and suggestions on the manuscript by J. A. Kashul.

Supplementary material

232_2014_9678_MOESM1_ESM.docx (22.1 mb)
Supplementary material 1 (DOCX 22653 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Aleksandr Sverzhinsky
    • 1
  • Shuo Qian
    • 2
  • Lin Yang
    • 3
  • Marc Allaire
    • 3
  • Isabel Moraes
    • 4
    • 5
    • 6
  • Dewang Ma
    • 7
  • Jacqueline W. Chung
    • 1
  • Manuela Zoonens
    • 8
    • 9
  • Jean-Luc Popot
    • 8
    • 9
  • James W. Coulton
    • 1
    • 10
    Email author
  1. 1.Department of Microbiology and ImmunologyMcGill UniversityMontrealCanada
  2. 2.Center for Structural Molecular Biology and Biology and Soft Matter DivisionOak Ridge National LaboratoryOak RidgeUSA
  3. 3.Photon Sciences DirectorateBrookhaven National LaboratoryUptonUSA
  4. 4.Membrane Protein LaboratoryDiamond Light SourceDidcotUK
  5. 5.Research Complex at Harwell Appleton Laboratory, Harwell Science and Innovation CampusDidcotUK
  6. 6.Department of Life SciencesImperial College LondonLondonUK
  7. 7.Faculté de Pharmacie and Département de ChimieUniversité de MontréalMontrealCanada
  8. 8.Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099ParisFrance
  9. 9.CNRS/Université Paris 7, Institut de Biologie Physico-Chimique (FRC 550)ParisFrance
  10. 10.Microbiome and Disease Tolerance CentreMontrealCanada

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