A Combined Spectroscopy and Computational Molecular Docking Investigation on the Coupling Between β-lactoglobulin Dimers and Vanillin

Protein − flavor interactions may influence flavor perception in food products and, therefore, they need to be deeply understood, at the molecular scale. We investigated the coupling between the bovine whey protein β-lactoglobulin (β-lg) and the vanillin (VAN), the main compound present in the flavor vanilla, at pH 7.1 (which is representative of close-to-neutral food formulations). Fluorescence quenching data showed that the static quenching mechanism was predominant in the β-lg/vanillin complexes formation, and allowed estimating its thermodynamic parameters Δ H V A N / β - l g = - 30.3 kJ · mol - 1 , Δ S V A N / β - l g = - 45.8 J · mol - 1 K - 1 , and Δ G V A N / β - l g ranging from - 16.9 to - 16.5 kJ · mol - 1 , showing that the β-lg-VAN complexation was spontaneous and enthalpically driven, with the involvement of both hydrogen bonding and hydrophobic interactions. In silico molecular docking assays were undertaken to identify the specific binding sites and to verify the nature of intermolecular interactions between β-lg and VAN. At pH 7.1, β-lg forms dimers in aqueous medium, and this important fact was considered. Molecular docking results showed that vanillin may bind to two possible sites: A-site (interface between the two β-lg monomers) and B-site (β-barrel entrance of β-lg). The conjoint analysis of in vitro and in silico findings indicated that the B-site is more likely to be the binding site of β-lg for the vanillin molecule. Indeed, while in the A-site hydrogen bonds seemed to be stronger, in the B-site there was a balance between the presence of hydrogen bonds, electrostatic interactions, and hydrophobic interactions, in particular involving aromatic amino acid residues. The β-lg amino acid residues involved in these interactions were Trp19, Tyr20, Val43, Glu44, Leu156, Glu157, and His161.