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.