title: Protein-lipid interactions with Fusobacterium nucleatum major outer membrane protein FomA: spin-label EPR and polarized infrared spectroscopy.
abstract: FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and lipid-protein interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a beta-sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane beta-barrel structure. IR dichroism of FomA indicates that the beta-strands are tilted by approximately 45 degrees relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry ( N b = 23 lipids/monomer) of lipid-protein interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric beta-barrel of 14-16 strands. The pronounced selectivity of interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8- to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.
authors.affiliation: Max-Planck-Institut fur biophysikalische Chemie, Abt. Spektroskopie, 37070 Gottingen, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Anbazhagan
authors.forename: N
authors.initials: N
authors.lastname: Vijay
authors.forename: J H
authors.initials: JH
authors.lastname: Kleinschmidt
authors.forename: D
authors.initials: D
authors.lastname: Marsh
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D016967:Fusobacterium nucleatum / Q000378:metabolism; D008563:Membrane Lipids / Q000032:analysis / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013055:Spectrophotometry, Infrared; D013113:Spin Labels; D013329:Structure-Activity Relationship
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008563:Membrane Lipids; D013113:Spin Labels; C083760:fomA protein, Fusobacterium