Probing the binding sites of resveratrol, genistein, and curcumin with milk β-lactoglobulin

Abstract

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of Kcurcumin–β-LG = 4.4 (± .4) × 104 M−1, Kresveratrol–β-LG = 4.2 (± .2) × 104 M−1, and Kgenistein–β-LG = 1.2 (± .2) × 104 M−1. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of −12.67 (curcumin–β-LG), −12.60 (resveratrol–β-LG), and −10.68 kcal/mol (genistein–β-LG). The order of binding was cur > res > gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.