title: NMR study of a membrane protein in detergent-free aqueous solution.
abstract: One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the beta-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed.
authors.affiliation: Unité Mixte de Recherche 7099, Centre National de la Recherche Scientifique (CNRS)/Université Paris-7, Institut de Biologie Physico-Chimique (CNRS FRC 550), 11 Rue Pierre et Marie Curie, F-75005 Paris, France.
authors.forename: Manuela
authors.initials: M
authors.lastname: Zoonens
authors.forename: Laurent J
authors.initials: LJ
authors.lastname: Catoire
authors.forename: Fabrice
authors.initials: F
authors.lastname: Giusti
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Popot
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011108:Polymers; D011487:Protein Conformation; D012996:Solutions; D014867:Water
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011108:Polymers; D012996:Solutions; D014867:Water; C078167:OMPA outer membrane proteins