Protein-lipid interactions with Fusobacterium nucleatum major outer membrane protein FomA: spin-label EPR and polarized infrared spectroscopy.
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.
Anbazhagan
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V
Max-Planck-Institut fur biophysikalische Chemie, Abt. Spektroskopie, 37070 Gottingen, Germany.
Vijay
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N
Kleinschmidt
J H
JH
Marsh
D
D
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
D001425:Bacterial Outer Membrane Proteins; D008563:Membrane Lipids; D013113:Spin Labels; C083760:fomA protein, Fusobacterium