Figure 3.

Modeling binding parameters from FCCS measurements. Fractional receptor occupancy can be derived from correlation amplitudes to derive (a) saturation- and (b) competition-binding isotherms. Cartoons show the binding interactions analyzed by FCCS. Fluorescent ligand (orange with red star) recognizes a lipid-embedded (violet) membrane receptor (blue). The receptor is fused to a functional tag (yellow) labeled with a fluorophore (green). The amplitudes are modeled as a function of titrating ligand concentration for receptor, ligand, and receptor-ligand complex. For saturation binding (a), correlation amplitude plots are shown for receptor (green, A_R), ligand (red, A_L), and complex (blue, A_X) as a function of fluorescent ligand concentrations at which ligand dissociation constants were set to 5 nM (yellow) or 0.5 nM (orange). Ligand concentration varied from 0.01 to 100 nM, and receptor concentration was kept constant at 0.5 nM. For competition-binding analysis (b), competitor concentration varied from 0.01 to 10,000 nM, and ligand and receptor concentrations were kept constant at 2 and 0.5 nM, respectively. Competition-binding isotherms can be derived by plotting A_X/A_L as a function of competitor concentration. Two different competition-binding cases were simulated in which ligand and receptor concentrations were kept constant while an unlabeled competitor concentration was titrated (b). The ligand and competitor affinities were assumed to be K_d = 0.5 nM, K_i = 0.5 nM (black) or K_d = 0.5 nM, K_i = 5 nM (cyan). A_R and A_L remain constant while A_X decreases with increasing competitor concentration.