Vibrational analysis of acetylcholine binding to the M₂ receptor

The M₂ muscarinic acetylcholine receptor (M₂R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M₂R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-¹³C labeled ACh, and assigned the bands at 1741 and 1246 cm^-1 as the C[double bond, length as m-dash]O and C-O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M₂R is close to that in aqueous solution (1736 cm^-1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778-1794 cm^-1), indicating a strong hydrogen bond, which probably formed with N404^6.52. We propose that a water molecule bridges ACh and N404^6.52. The other ACh terminal is positively charged, and it interacts with negatively charged D103^3.32. The present study revealed that D103^3.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D103^3.32 in the free M₂R.