Fluorescence spectroscopy, 3D-QSAR, and molecular dynamics analyses reveal the interaction mechanisms of flavonoids...

This research elucidates the mechanisms of non-covalent interactions between flavonoids and lysozyme. Binding constants were determined via fluorescence spectroscopy, and a Topomer CoMFA 3D-QSAR model was developed, effectively explaining the binding behavior. Detailed studies on rutin, naringenin, and hesperetin showed binding occurred via a static quenching mechanism with a 1:1 stoichiometry, and hydrogen bonding and van der Waals forces were indicated as the primary drivers. Synchronous fluorescence revealed flavonoid binding disrupted the tryptophan microenvironment but left tyrosine relatively stable. IGM analysis of rutin glycosylation revealed a rigid intramolecular hydrogen-bond network, whereas the aglycones were less stable. Intermolecularly, rutin formed multiple hydrogen bonds and extensive hydrophobic interactions, while aglycone binding was simpler. MD simulations and free energy calculations yielded the following binding affinity: hesperetin > naringenin > rutin, underscoring the importance of solvation effects. This work provides molecular-level insights into the structure-activity relationship, aiding the design of flavonoid delivery systems.