Glycosylation reshapes coumarin recognition by β-lactoglobulin: Polar-contact-solvation trade-off revealed by multispectroscopy and molecular simulation.

Ligand glycosylation is often assumed to enhance protein binding by increasing hydrogen-bonding capacity, but its actual role in coumarin-β-lactoglobulin (β-LG) recognition has not been clearly resolved. Here, fraxin (FXN) and its aglycone fraxetin (FXT) were used as a paired model to examine how a glucose moiety regulates β-LG binding through multispectroscopic analysis and molecular simulations. Both ligands associated with β-LG and quenched its intrinsic fluorescence, but FXT showed stronger quenching, higher apparent affinity, a shorter FRET-derived donor-acceptor distance, and a more pronounced reduction in fluorescence lifetime. Thermodynamic analysis indicated spontaneous, entropy-favored binding for both systems. Synchronous and three-dimensional fluorescence spectra further showed that FXT caused greater perturbation of the aromatic-residue microenvironment, whereas far-UV CD revealed only limited secondary-structure fluctuations without a consistent concentration-dependent trend. Docking, MD simulation, and MM/PBSA calculations indicated that the glucose group in FXN introduced additional polar contacts but also a larger polar-solvation penalty and possible steric constraints. In contrast, FXT achieved closer hydrophobic accommodation near the fluorescence-sensitive region of β-LG. These results suggest that glycosylation does not simply strengthen coumarin-β-LG binding, but reshapes molecular recognition through a trade-off among polar interactions, desolvation, and hydrophobic fitting.