Association of lipids with milk α- and β-caseins

We report the molecular interaction and the binding sites of cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) with milk α- and β-caseins in aquous solution at physiological conditions. Fourier transform infrared (FTIR), fluorescence spectroscopic methods and molecular modeling were used to determine the binding sites of lipid–protein complexes and the effect of lipid interaction on the stability and conformation of α- and β-caseins. Structural analysis showed that lipids bind casein via mainly hydrophobic contact with association constants of KCHOL-α-casein = 1.0 (±0.1) × 104 M−1, KDOPE-α-casein = 5.0 (±0.07) × 103 M−1, KDDAB-α-casein = 2.0 (±0.06) × 104 M−1, KDOTAP-α-casein = 1.5 (±0.6) × 104 M−1, KCHOL-β-casein = 1.0 (± 0.3) × 104 M−1, KDOPE-β-casein = 1.5 (±0.06) × 103 M−1, KDDAB-β-casein = 1.7 (±0.3) × 104 M−1 and KDOTAP-β-casein = 2.1 (±0.5) × 104 M−1. The average number of binding sites occupied by lipid molecules on protein (n) were from 0.7 to 1.1. Docking showed different binding sites for α- and β-caseins toward lipid complexation with the free binding energies from −10 to −13 kcal/mol. Casein conformation was altered by lipid interaction with a reduction of α-helix and β-sheet and an increase of random coil and turn structure suggesting a partial protein unfolding.Abbreviations: Cascasein; CHOLcholesterol; DOTAP1,2-dioleoyl-3-trimethylammonium-propane; DDABdioctadecyldimethylammonium bromide; DOPEdioleoylphosphatidylethanolamine; FTIRFourier transform infrared spectroscopy; CDcircular dichroism