Figure 2
Functional characterization of SMA copolymer solubilized α1-His GlyR. (a) Example trace of primary thermophoresis data. Thermophoretic movement of α1-His GlyR nanodiscs is expressed as the change in fluorescence signal between initial fluorescence Fcold (0 s) and fluorescence after thermodiffusion Fhot (15 s) and was calculated as ratio of both values as described in the “Materials and Methods” section. Inset shows an amplification of representative fluorescence traces of the thermophoretic movement of the fluorescence labeled α1-His GlyR between 14 and 15 s (Fhot) obtained at different glycine concentrations [0 and 1 (black), 10 and 100 (gray) and 1000 and 3000 µM glycine (red)]. (b) The change in thermophoretic movement upon binding of increasing concentrations of Gly results in a change of the relative fluorescence between the unbound state (black) and glycine-bound state (red) after 15 s. (c) Initial fluorescence count distribution for each concentration is under 10% and showing no ligand-dependent fluorescence quenching. (d) Dose–response curve obtained from MST experiments of α1-His GlyR. Binding of glycine to fluorescence-labeled α1-GlyR was obtained with a titration series from 3 mM to 0.73 µM in PBS buffer, pH 7.4. The change in thermophoretic signal leads to a cEC50 of 65 ± 22.8 µM. Error bars represent SEM between n = 3 independent experiments.
