title: FhuA interactions in a detergent-free nanodisc environment.
abstract: TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study β-barrel membrane proteins in a membrane-like environment.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada; Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Allan
authors.initials: A
authors.lastname: Mills
authors.forename: Hai-Tuong
authors.initials: HT
authors.lastname: Le
authors.forename: James W
authors.initials: JW
authors.lastname: Coulton
authors.forename: Franck
authors.initials: F
authors.lastname: Duong
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003087:Colicins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005291:Ferrichrome / Q000031:analogs & derivatives* / Q000737:chemistry; D015870:Gene Expression; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D018716:Molecular Mimicry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; D005291:Ferrichrome; C021678:ferricrocin
keywords: BN; CN; Channels; DDM; Fc; ITC; LDAO; MSP; Membrane; N-lauryldimethyl amine-N-oxide; Nanodiscs; Siderophore; Transporters; blue-native; clear-native; dodecyl-β-d-maltoside; ferricrocin; isothermal titration calorimetry; membrane scaffold protein; β-Barrel proteins