Ion channel proteins can be in vitro translated into nanoscale lipid bilayers known as nanodiscs. Winterstein et al. show that they can subsequently insert into planar bilayers, providing a rapid and contamination-free method for functional characterization.

Fibronectin domain monobodies bind to both sides of Fluc Fl− channels in a negatively cooperative way, but crystal structures show two monobodies binding simultaneously. Turman and Stockbridge resolve this contradiction by showing that monobodies block channel pores by means of a negatively charged loop.

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 monobodybinding 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.ion channel | monobody | block | cooperativity S everal years ago, Baker et al.(1) discovered that many microorganisms harbor in their membranes anion-exporter proteins that keep cytoplasmic F − below the toxic concentrations encountered throughout the aqueous environment. The novelty of this previously unsuspected microbial physiology is mirrored in the unusual molecular architecture of one class of these exporters, the Fluc family. Flucs are highly F − -selective ion channels built as dual-topology homodimers, wherein the paired subunits of the functional channel assemble in antiparallel transmembrane topology (2, 3). This architecture demands that, if the twin subunits adopt identical conformations, the channel must present structurally identical ion entryways to the two sides of the membrane, in sharp contrast to the parallel assembly of conventional multisubunit membrane proteins. Antiparallel assembly of Flucs was established definitively (2) by the use of "monobodies," small fibronectindomain proteins of known structure engineered by random variation of amino acid sequences and selected in combinatorial libraries as nanomolar affinity-specific binders (4). In double-sided perfusion experiments, single Fluc channels were shown to be blocked with similar kinetics by monobodies added separately to the internal or external aqueous solution. The channel thus presents to each side of the membrane identical epitopes for the blocker, as required of symmetrical, antiparallel assembly (Fig. 1A), a circumstance that naturally raises the question can monobodies occupy both blocking sites simultaneously?We address this question by direct binding and single-channel experiments with monobody on both sides of the channel. The results reveal a previously unobserved "trimolecular" channelblocking beha...