Binding parameters and molecular dynamics of β-lactoglobulin-vanillic acid complexation as a function of pH - Part A: Acidic pH

Protein-phenolic compound interactions are commonly investigated with inappropriate linear equations for the analysis of binding strength and stoichiometry. This work utilises more appropriate protocols for the investigation of molecular interactions between vanillic acid and beta-lactoglobulin at pH 2.4, where the protein predominately exists as a monomer. Non-linear binding and Job plot analysis were conducted on fluorescence data to effectively determine the interaction's dissociation constant (K-D, 2.93 x 10(-5) M) and stoichiometry (1:1). Furthermore, spectroscopic techniques revealed statistically significant alterations to the conformational characteristics of beta-lactoglobulin upon complexation. Molecular dynamics (MD) simulations support a 1:1 interaction stoichiometry and reveal that the stabilisation of vanillic acid was dynamic in nature but mainly supported by four pi-alkyl interactions and one hydrogen bond, located within the beta-barrel of the monomer. Water molecules, which are generally not accounted for in MD simulation analysis, were shown to be an important factor in the ligand stabilization via bridging interactions.

Automated summary

This study investigates the molecular interactions between vanillic acid and beta-lactoglobulin using appropriate protocols, determining the dissociation constant and stoichiometry, and revealing significant alterations to the conformational characteristics of the protein upon complexation. Molecular dynamics simulations support a 1:1 interaction stoichiometry and highlight the dynamic nature of vanillic acid stabilization, primarily through pi-alkyl interactions and hydrogen bonding. Water molecules play a crucial role in ligand stabilization through bridging interactions, which are often overlooked in MD simulation analysis.