High-Resolution Structure of a Membrane Protein Transferred from Amphipol to a Lipidic Mesophase
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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.
KeywordsAmphipol Membrane protein crystallization Bacteriorhodopsin Monoolein In meso crystallization
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.
- Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221CrossRefGoogle Scholar
- Banères JL, Popot JL, Mouillac B (2011) New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322Google Scholar
- Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Banères JL, Durand G, Zito F, Pucci B, Popot J-L (2012) Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance. Biochemistry 51:1416–1430CrossRefGoogle Scholar
- Damian M, Marie J, Leyris J-P, Fehrentz J-A, Verdié P, Martinez J, Banères J-L, Mary S (2012) High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs. J Biol Chem 287:3630–3641CrossRefGoogle Scholar
- Giusti F, Popot J-L, Tribet C (2012) Well-defined critical association concentration and rapid adsorption at the air/water interface of a short amphiphilic polymer, amphipol A8-35: a study by Förster resonance energy transfer and dynamic surface tension measurements. Langmuir 28:10372–10380CrossRefGoogle Scholar
- Kleinschmidt JH, Popot J-L (2014) Folding and stability of integral membrane proteins in amphipols. Arch Biochem Biophys (in press)Google Scholar
- Marie E, Sagan S, Cribier S, Tribet C (2014). Amphiphilic macromolecules on cell membranes: from protective layers to controlled permeabilization. J Membr BiolGoogle Scholar
- Popot J-L, Berry EA, Charvolin D, Creuzenet C, Ebel C, Engelman DM, Flötenmeyer M, Giusti F, Gohon Y, 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 60:1559–1574CrossRefGoogle Scholar
- Popot J-L, Althoff T, Bagnard D, Banères J-L, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408CrossRefGoogle Scholar
- Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67:235–242CrossRefGoogle Scholar