title: Amphipol-trapped ExbB-ExbD membrane protein complex from Escherichia coli: a biochemical and structural case study.
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.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, H3A 2B4, QC, Canada.
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Sverzhinsky
authors.forename: Shuo
authors.initials: S
authors.lastname: Qian
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Marc
authors.initials: M
authors.lastname: Allaire
authors.forename: Isabel
authors.initials: I
authors.lastname: Moraes
authors.forename: Dewang
authors.initials: D
authors.lastname: Ma
authors.forename: Jacqueline W
authors.initials: JW
authors.lastname: Chung
authors.forename: Manuela
authors.initials: M
authors.lastname: Zoonens
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Popot
authors.forename: James W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D057927:Hydrophobic and Hydrophilic Interactions; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D011108:Polymers / Q000737:chemistry*; D011437:Propylamines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; D046912:Multiprotein Complexes; D011108:Polymers; D011437:Propylamines; D013501:Surface-Active Agents; C499017:amphipol A8-35; C060790:exbD protein, E coli