Fig. 2. LicB displays promiscuous activity toward choline-like molecules.

(A) SSM electrophysiology recordings of choline transport in LicB proteoliposomes. Currents recorded between 1 and 1.3 s after addition of choline are shown in blue. Recordings with protein-free liposomes in the presence of 5 mM choline is shown in red. (B) Determination of EC₅₀ values for choline, arsenocholine, and acetylcholine based on SSM recording as shown in (A) and fig. S2. (C) WTA and LTA extraction from S. pneumoniae cells grown in the presence of [³H]-acetylcholine radioactively labeled at different positions indicated by dotted circles. Histograms show the [³H] radioactivity from extracted WTA and LTA. NC, negative control performed with nonradioactive acetylcholine. (D) Normalized amplitudes of SSM currents measured in the presence of sybodies A to L (500 nM), under 5 mM choline transport conditions based on data shown in fig. S3. The asterisk denotes sybody C, which was used for structural studies. n = 3 to 4 biological replicates; n = 2 to 3 technical replicates, for all experiments.