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Two-sided block of a dual-topology F- channel.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2015 May 05; Vol. 112 (18), pp. 5697-701. Date of Electronic Publication: 2015 Apr 20. - Publication Year :
- 2015
-
Abstract
- The Fluc family is a set of small membrane proteins forming F(-)-specific electrodiffusive ion channels that rescue microorganisms from F(-) toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.
- Subjects :
- Anisotropy
Antibodies, Monoclonal chemistry
Binding Sites
Cysteine chemistry
Dose-Response Relationship, Drug
Epitopes chemistry
Fluorescence Polarization
Kinetics
Lipid Bilayers chemistry
Microscopy, Fluorescence
Models, Theoretical
Mutation
Naphthalenesulfonates chemistry
Protein Binding
Static Electricity
Bacterial Proteins metabolism
Fluorine chemistry
Ion Channels chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 112
- Issue :
- 18
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 25902543
- Full Text :
- https://doi.org/10.1073/pnas.1505301112