Exploring the binding mechanism of folic acid-phosvitin complexes and the effects of ultrasound-pH treatment on their properties: Kinetic, multispectral, and dynamic binding behavior analyses.

Understanding protein-bioactive molecule interactions and their precise modulation via physical field processing is fundamental to developing functional food ingredients. This study investigated the structural reorganization and dynamic binding behavior of folic acid (FA)-phosvitin (PSV) complexes under ultrasound-pH synergistic treatment (0-600 W, pH 12.0). A comprehensive approach combining multispectral analysis, thermodynamic evaluation, and molecular dynamics (MD) simulations was employed to elucidate the underlying assembly mechanisms. Experimental and simulation results confirmed that FA bound to PSV via a spontaneous static quenching mechanism, primarily driven by hydrogen bonding and van der Waals forces. MD simulations (100 ns) visualized an induced-fit binding process, where FA was stably anchored within the PSV hydrophobic cavity by key residues (ARG150, ASP70, and SER148) with a binding energy of -26.63 kcal/mol, leading to reduced residue flexibility and a more compact tertiary conformation. Crucially, two-dimensional correlation spectroscopy (2D-COS) analysis unveiled a sequential conformational transition mechanism orchestrated by ultrasound: the disruption of intermolecular hydrogen bonds and α-helices preceded the rearrangement of β-sheets and hydrophobic exposure. Raman spectroscopy further highlighted a specific disulfide bond transition from intramolecular (gauche-gauche-gauche) to intermolecular (trans-gauche-trans) configurations, indicating that ultrasound-pH treatment promoted the formation of a stable, cross-linked intermolecular network. This structural remodeling, optimized at 450 W, significantly enhanced the functional properties of the complexes, yielding a surface hydrophobicity of 87.50%, an emulsifying activity index (EAI) of 112.36 m2/g, and a foaming capacity of 209.48%. Furthermore, the complexes exhibited superior antioxidant activity (DPPH: 62.96%; ABTS: 79.21%) compared to native PSV due to the modified exposure of active groups. In conclusion, this study provides deep theoretical insights into the ultrasound-modulated assembly mechanism and the structure-function relationship of FA-PSV complexes, supporting their potential application as multifunctional ingredients.