Deciphering the Stevioside-Whey Protein Binding Mechanism: A Synergistic Multispectral and Computational Docking...

This study integrates multispectral characterization with computational modeling to elucidate the molecular mechanisms underlying stevioside (STE) and whey protein (WP) complexation. Using UV-visible absorption and fluorescence spectroscopy, we confirm the formation of STE-WP complexes via static quenching, as supported by Stern-Volmer constants. Thermodynamic analysis reveals an entropy-driven binding process (ΔS = +976.7 J·mol^-1·K^-1), where the favorable entropy change outweighs the unfavorable enthalpy change (ΔH = +275.2 kJ·mol^-1), resulting in spontaneous association (ΔG = -20.7 kJ·mol^-1). Secondary structural changes induced by STE binding were quantified through circular dichroism deconvolution, showing a 153% increase in α-helix content (16% → 40.5%), accompanied by decreases in β-sheet (10% → 3%) and β-turn (67.1% → 41.2%) content, as well as an increase in random coil structure (6.8% → 15.7%). Molecular docking and molecular dynamics simulations reveal that STE predominantly localizes within the hydrophobic pockets of WP. The stabilized binding interaction is mediated by a network of hydrogen bonds and hydrophobic interactions involving multiple key amino acid residues. These findings establish a structure-energy framework that elucidates the thermodynamic driving forces and binding mechanisms underlying the co-assembly of STE and WP, thereby providing a theoretical foundation for their application in functional foods.