Fig. 3: Dual Interaction Sequential Compound (DISCo)-BRET measures simultaneous binding of tracer ligands to two distinct FFA1 binding sites. | Nature Communications

Fig. 3: Dual Interaction Sequential Compound (DISCo)-BRET measures simultaneous binding of tracer ligands to two distinct FFA1 binding sites.

From: Multicolored sequential resonance energy transfer for detection of simultaneous ligand binding at G protein-coupled receptors

Fig. 3

A The spectral properties of Nluc, TUG-2591 and TUG-2597 are shown. Spectral overlap in Nluc emission with TUG-2597 excitation, as well as overlap in TUG-2597 emission with TUG-2591 excitation are highlighted. B A cartoon diagram demonstrates predicted BRET energy transfer between Nluc, a red site one tracer and a green site two tracer. The cartoons depict the experimental conditions used incubating Nluc-FFA1 or FFA1-Nluc cells with: i) the red site one tracer alone; ii) the red site one tracer combined with a competing ligand; iii) the red site one tracer and the green site two tracer; and iv) the red site one tracer, the green site two tracer and a competing ligand for site one. C Luminescent emission spectra were recorded from cells expressing an N terminally Nluc tagged FFA1 with increasing concentrations of the red tracer TUG-2591. All spectra are shown as ‘specific BRET’ by subtracting the spectrum obtained in the presence competing ligands for both site one and two, TUG-905 (10 μΜ) and T360 (10 μΜ). Data are shown for the control condition (Ci); for the control non-specific (NS) condition, in the presence of TUG-770 to show non-specific TUG-2591 binding (Cii); in the presence of 316 nM of the green tracer TUG-2597 (Ciii); or non-specific binding in the presence of TUG-2597, with both 316 nM TUG-2597 and 10 μΜ TUG-905 (Civ). D Comparable experiments to those presented in C were conducted using cells expressing an FFA1 construct with Nluc fused to the C terminal of the receptor. DISCo-BRET saturation binding curves showing total and non-specific (in the presence of 10 μM TUG-905) TUG-2591 binding in the presence of TUG-2597 at either N (E) or C (F) terminal Nluc tagged FFA1 constructs are shown. DISCo-BRET was calculated by taking the area under the SulfoCy5 peaks (660-730 nm) divided by the area under the NBD peaks (490-560 nm) from the specific BRET spectra shown in (C and D). All DISCo-BRET data shown are mean ± SEM from N = 4 independent experiments. Source data are provided as a Source Data file.

Back to article page