Multi-spectroscopic, conformational, and computational atomic-level insights into the interaction of β-lactoglobulin with apigenin at different pH levels

Abstract

β-Lactoglobulin commonly undergoes conformational changes at different pH levels (e.g., Tanford transition above pH ~7 and dimer-to-monomer dissociation below pH 3.0), which affects its ligand-binding properties. In this study, multi-spectroscopic methods, conformational characterization, and molecular simulation provided insight into the mechanism of β-lactoglobulin interaction with apigenin at various pH levels (2.6, 6.2, 7.1, and 8.2). Initially, apigenin was found to statically quench the intrinsic fluorescence of β-lactoglobulin at all pH levels, demonstrating their successful interaction. The affinity decreased in the order of pH 6.2 > 8.2 > 7.1 > 2.6. At pH 7.1 and 8.2, the hydrophobic interactions between β-lactoglobulin and apigenin were predominant, while at pH 2.6 and 6.2, the main interactions between β-lactoglobulin and apigenin were van der Waals forces and hydrogen bonding. Molecular docking showed that apigenin preferred to bind to the outer surface of β-lactoglobulin with a closed β-barrel (pH 2.6 and 6.2), in contrast to the open β-barrel at pH 7.1 and 8.2, where a large number of non-polar residues were distributed and apigenin could be inserted therein. In the case of the strongest affinity at pH 6.2, the Trp19, Val43, and His161 residues were found to significantly contribute to the binding free energy precisely because of their hydrogen bonding with apigenin. Moreover, the conformational study of β-lactoglobulin with and without apigenin showed an enhanced solvent accessibility around Trp residues, and a decrease in surface hydrophobicity with an indistinctive unfolding. The above results demonstrated the slight increase in β-lactoglobulin size without significant aggregation.