Infrared Spectroscopy of Amide Modes in Peptides: Experimental FTIR and QM/MM Protocol Insights

This study presents a combined experimental and theoretical investigation of the infrared vibrational spectrum of human serum albumin (HSA) complexed with the ligand warfarin. Experimental spectra in the 1500–1700 cm region reveal that the amide CO vibrational bands of HSA remain essentially unchanged in the presence of warfarin's carbonyl groups, indicating limited coupling between protein and ligand vibrational modes. Complementary QM/MM harmonic frequency calculations using a cluster model comprising six amino acids plus warfarin predict the carbonyl stretching region between 1550 and 1750 cm, showing only minor intensity changes together with a slight red shift upon ligand binding. These subtle spectral shifts suggest localized structural and electronic perturbations near the binding site without major alterations to the protein backbone environment. The study highlights the importance of combining experimental IR spectroscopy (FTIR) and refined quantum chemical modeling to elucidate protein–ligand interactions at the molecular vibrational level. Potential improvements in theoretical modeling to better align with experiment are discussed, including the use of larger cluster models, explicit solvation, and anharmonic corrections.