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. 2016 Jul 23;5:e18767. doi: 10.7554/eLife.18767

Figure 2. Proper assembly of fused-domain Ec2.

(a) Cartoon of fused-domain construct, showing numbered transmembrane helices in Ec2 domains (open) fused by an 'inversion linker' containing a non-dimerizing glycophorin A helix (grey). (b) Coomassie-stained SDS PAGE gel of purified Ec2 homodimer (left lane) and concatemer (right lane), with soluble-marker ladder positions indicated. (c) Representative F efflux traces of indicated concatemeric constructs. (d) Representative single-channel recordings in the presence of 75–150 nM monobody, which induces long-lived nonconducting 'blocked' intervals; conducting intervals in these traces represent times when channels are free of monobody, with intrinsic open probability >95% (Stockbridge et al., 2014). Grey lines represent blocked current level.

DOI: http://dx.doi.org/10.7554/eLife.18767.005

Figure 2.

Figure 2—figure supplement 1. Amino acid sequence of WT/WT Ec2 concatemer.

Figure 2—figure supplement 1.

Fluc helices are indicated by red residues, and underlined residues indicate the artificial inversion linker, which includes a nondimerizing glycophorin A helix (orange residues). Lower case characters indicate the C-terminal His6 affinity tag and linker.
Figure 2—figure supplement 2. Concatemeric Ec2 retains F selectivity.

Figure 2—figure supplement 2.

Anion efflux traces shown for WT/WT concatemer, using liposomes loaded with 150 mM KF + 150 mM KCl. Efflux of the different ions was followed by F- or Cl--selective electrodes.
Figure 2—figure supplement 3. Monobody block to baseline.

Figure 2—figure supplement 3.

Single-channel traces of the WT/WT Ec2 concatemer here are used to demonstrate that monobody fully blocks the concatemer. Planar bilayers were formed and voltage was set to 200 mV. Recordings were begun in the absence of monobody to catch the first channel insertion into the bilayer (arrow). Initial monobody-free measurements show the high open probability of the channel. At the start of the open bar, monobody (200 nM) was added to the solution without stirring, and discrete monobody blocks commenced after the reagent diffused into solution. The level of current before channel insertion is indistinguishable from the level of monobody block, observed over at least four such experiments.