title: Proof of dual-topology architecture of Fluc F- channels with monobody blockers.
abstract: Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in their quaternary architecture: they are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here, we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion manoeuvre, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA.
authors.forename: Randy B
authors.initials: RB
authors.lastname: Stockbridge
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
mesh_terms: D005459:Fluorides / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D020836:Protein Structure, Quaternary
chemical_list: D007473:Ion Channels; D008051:Lipid Bilayers; D005459:Fluorides