The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 997–1004 | Cite as

High-Resolution Structure of a Membrane Protein Transferred from Amphipol to a Lipidic Mesophase

  • V. Polovinkin
  • I. Gushchin
  • M. Sintsov
  • E. Round
  • T. Balandin
  • P. Chervakov
  • V. Schevchenko
  • P. Utrobin
  • A. Popov
  • V. Borshchevskiy
  • A. Mishin
  • A. Kuklin
  • D. Willbold
  • V. Chupin
  • J.-L. Popot
  • V. Gordeliy
Article

Abstract

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.

Keywords

Amphipol Membrane protein crystallization Bacteriorhodopsin Monoolein In meso crystallization 

Notes

Acknowledgments

Particular thanks are due to Fabrice Giusti (UMR 7099) for synthesizing the amphipols used in the present work. The diffraction experiments were performed at the beamline ID23-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to the ESRF beamline staff for assistance. This work was supported by the program “Chaires d’excellence, édition 2008’’ of the Agence Nationale de la Recherche France, by the Commissariat à l’Énergie Atomique (Institut de Biologie Structurale), by the Helmholtz Gemeinschaft (Research Centre Jülich) Special Topic of Cooperation 5.1 specific agreement, by a Marie Curie grant (Seventh Framework Programme-PEOPLE-2007-1-1-Initial Training Networks, project Structural Biology of Membrane Proteins), by a European Commission Seventh Framework Programme grant for the European Drug Initiative on Channels and Transporters consortium (HEALTH-201924), by the Centre National pour la Recherche Scientifique, by University Paris–7, and by the “Initiative d’Excellence” program of the French State (Grant “DYNAMO”, ANR-11-LABX-0011-01). Vitaly Polovinkin is deeply thankful to the Fondation Nanosciences for financial support. Part of this work was supported by the German Ministry of Education and Research (PhoNa-Photonic Nanomaterials). Protein expression, crystallization experiments and data treatment were supported by Russian Scientific Foundation (project 14-14-00995). We acknowledge support of this work by the Russian Foundation for Basic Research (Research project 13-04-01700), by the Russian program “5Top100” and by the Ministry of Education and Science of the Russian Federation. This work was supported by ONEXIM, Russia.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • V. Polovinkin
    • 1
    • 2
    • 3
    • 4
  • I. Gushchin
    • 1
    • 2
    • 3
    • 4
  • M. Sintsov
    • 4
  • E. Round
    • 5
  • T. Balandin
    • 5
  • P. Chervakov
    • 5
  • V. Schevchenko
    • 5
  • P. Utrobin
    • 1
    • 2
    • 3
  • A. Popov
    • 6
  • V. Borshchevskiy
    • 4
    • 5
  • A. Mishin
    • 4
  • A. Kuklin
    • 7
  • D. Willbold
    • 5
    • 8
  • V. Chupin
    • 4
  • J.-L. Popot
    • 9
  • V. Gordeliy
    • 1
    • 2
    • 3
    • 4
    • 5
  1. 1.Univ. Grenoble Alpes, IBSGrenobleFrance
  2. 2.CNRS, IBSGrenobleFrance
  3. 3.CEA, IBSGrenobleFrance
  4. 4.Laboratory for Advanced Studies of Membrane ProteinsMoscow Institute of Physics and TechnologyDolgoprudnyRussia
  5. 5.Institute of Complex Systems (ICS), ICS-6: Structural BiochemistryResearch Centre JuelichJuelichGermany
  6. 6.European Synchrotron Radiation FacilityGrenobleFrance
  7. 7.Frank Laboratory of Neutron PhysicsJoint Institute for Nuclear ResearchDubnaRussia
  8. 8.Institut für Physikalische BiologieHeinrich-Heine-Universität DüsseldorfDüsseldorfGermany
  9. 9.Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099, Institut de Biologie Physico-Chimique (CNRS FRC 550)Centre National de la Recherche Scientifique and Université Paris-7ParisFrance

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