Deciphering the non-covalent binding patterns of three whey proteins with rosmarinic acid by multi-spectroscopic, molecular docking and molecular dynamics simulation approaches

Binding of rosmarinic acid (RA) with three whey proteins, β-lactoglobulin (β-LG), α-lactalbumin (α-LA) and bovine serum album (BSA) was deciphered in the present study. Steady and time-resolved fluorescence spectra revealed the occurrence of fluorescence quenching via static type with binding constant sequence of BSA > α-LA > β-LG. Thermodynamic parameters indicated the dominated driving force of hydrophobic effect. Synchronous and three-dimensional fluorescence spectra confirmed the alteration in hydrophobic microenvironment of TRP. DSC and FT-IR spectra indicated the RA was successfully encapsulated in the complex. No obvious impact of RA binding on the secondary structure of whey proteins was observed by CD spectra. Molecular docking indicated that RA bound in calyx of β-LG, cleft entrance between α-helical and β-sheet-rich domains of α-LA and Sudlow's site I of BSA with binding affinity of BSA > α-LA > β-LG on basis of hydrophobic force and hydrogen bonds. Molecular dynamics simulation was conducted and RMSF calculations revealed that the fluctuation of interacting residues between RA and proteins becomes restricted to a certain extent. More hydrogen bonds were involved in binding between β-LG and RA. Molecular Mechanics Generalized Born Surface Area (MMGBSA) calculations suggested that β-LG/RA complex was more stable with major contribution from electrostatic interactions. Data may provide useful information for further investigation about fabrication of RA protecting system and development of whey protein-RA functional materials. • Binding constant with RA was in the sequence of BSA > α-LA > β-LG. • The dominated driving force for binding of RA with whey proteins was hydrophobic effect. • DSC and FT-IR spectra indicated that RA was successfully encapsulated in protein after complexation. • RA bound in calyx of β-LG, cleft entrance between α-helical and β-sheet-rich domains of α-LA and Sudlow's site I of BSA. • Molecular dynamics simulation indicated that β-LG/RA complex was more stable than α-LA/RA and BSA/RA.