Elucidating binding mechanisms of caffeic acid and resveratrol by beta-lactoglobulin: Insights into hydrophobic interactions and complex formation

Employing beta-lactoglobulin (beta-Lg) as a bioactive carrier substantially enhances the stability of small molecule active compounds. This research examines the intricate interaction mechanisms between beta-Lg and caffeic acid (CA) as well as resveratrol (RES). Utilizing UV and fluorescence spectral analysis, it was discovered that both CA and RES exhibit a notable fluorescence quenching effect on beta-Lg, predominantly characterized by static quenching. Thermodynamic assessments revealed that hydrophobic interactions primarily drive the formation of complexes between the two small molecules and beta-Lg. Concerning binding affinity, beta-Lg/CA displayed a stronger association compared to beta-Lg/RES. Synchronous fluorescence spectroscopy outcomes demonstrated alterations in the hydrophobic microenvironment of aromatic amino acids. Particle size evaluations indicated a reduction in the size of both complexes, suggesting that CA and RES effectively enhance the dispersion of complexes within the solution. Molecular docking and molecular dynamics simulations substantiated that upon binding with proteins, small molecules remain stably situated within the hydrophobic cavity of beta-Lg, without considerable secondary structure modifications. Based on the MM/PBSA method, the energy contribution distribution of amino acid residues disclosed that ILE56, PHE105, and 58LEU contribute maxima beta y to beta-Lg/CA, while MET107, ILE84, and VAL41 contribute most significantly to beta-Lg/RES. Regarding antioxidant activity, the following hierarchy was observed: beta-Lg/RES > beta-Lg/CA > RES > CA. The insights gained from this investigation offer invaluable information for developing polyphenol protection systems and designing advanced beta-lactoglobulin carriers.