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Proof of dual-topology architecture of Fluc F− channels with monobody blockers

Author

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  • Randy B. Stockbridge

    (Howard Hughes Medical Institute, Brandeis University)

  • Akiko Koide

    (University of Chicago)

  • Christopher Miller

    (Howard Hughes Medical Institute, Brandeis University)

  • Shohei Koide

    (University of Chicago)

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.

Suggested Citation

  • Randy B. Stockbridge & Akiko Koide & Christopher Miller & Shohei Koide, 2014. "Proof of dual-topology architecture of Fluc F− channels with monobody blockers," Nature Communications, Nature, vol. 5(1), pages 1-5, December.
  • Handle: RePEc:nat:natcom:v:5:y:2014:i:1:d:10.1038_ncomms6120
    DOI: 10.1038/ncomms6120
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    Cited by:

    1. Randy B. Stockbridge & Lawrence P. Wackett, 2024. "The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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