Fig. 5: DISCo-BRET kinetics reveals a slow off rate at site one when a compound is bound to site two. | Nature Communications

Fig. 5: DISCo-BRET kinetics reveals a slow off rate at site one when a compound is bound to site two.

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

Fig. 5

A A cartoon diagram depicting a DISCo-BRET dissociation-rate experiment. Receptor was pre bound to both the red and green tracers, followed by addition of a competing ligands for both site one and site two (left), site one alone (middle), or site two alone (right). Experiments were conducted where cells were first treated with TUG-2591 (100 nM) and TUG-2597 (316 nM) together, before the addition (marked as Dis.) of TUG-905 (10 μΜ), T360 (10 μM) or a combination of TUG-905 and T360 (10 μM each) to assess the dissociation of the FFA1/ TUG-2591/ TUG-2597 complex. Data from these experiments are shown as the 690 nm / 540 nm (B), or 540 nm / 450 nm (C) luminescent emission ratios. D The dissociation portion of the curve from (B) is plotted and fit to a one site exponential decay model. E The dissociation portion of the curve from (C) is plotted and fit to a one site exponential decay model. Data in (B–E) are from N = 3 independent experiments. Dissociation NanoBRET experiments are shown for TUG-2597 (F) and TUG-2591 (G), following treatment with T360 (10 μM) and TUG-905 (10 μM) respectively. NanoBRET data are mean ± SEM from from N = 3 (F) or N = 4 (G) independent experiments. Source data are provided as a Source Data file.

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