Methods for quantifying ligand efficacy in G-protein coupled receptors using single-molecule fluorescence energy transfer

A method for determining a molecular efficacy of a ligand, the method comprising:

• obtaining a labelled beta-2 adrenergic receptor (β2AR) and contacting the labelled β2AR with the ligand;
• detecting an effective rate of generating Gs(GTP) from Gs(GDP) provided by the ligand (“ligand effective rate”);
• and
• detecting an apparent EC50 for GDP binding of the ligand (“ligand EC50”);
• wherein
• η1 is the ligand effective rate normalized to an effective rate of adrenaline in generating Gs(GTP) from Gs(GDP);
• η2 is the ligand EC50 normalized to an apparent EC50 for GDP binding of adrenaline;
• and
• a value greater than 1.0 for the product of η1 and η2 indicates the ligand is more efficacious than adrenaline.