AbstractThe intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M₂ muscarinic acetylcholine receptor (M₂R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M₂R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M₂R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.