Binding of Cationic Lipids to Milk β-Lactoglobulin

We determined the bindings of several lipids such as cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to beta-lactoglobulin (beta-LG) at physiological conditions. FTIR, CD, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of lipid-protein complexes. Structural analysis showed that lipids bind beta-LG via both hydrophilic and hydrophobic interactions with overall binding constants of KCHOL-beta-LG = 6.0 (+/- 0.6) X 10(3) M-1, KDOPE-beta-LG = 6.5 (+/- 0.7) x 10(3) M-1 KDDAB-beta-LG = 1.6 (+/- 0.3) x 10(4) M-1, and KDOTAP-beta-LG = 2.2 (+/- 0.67) X 10(4) M-1. The number of lipid bound per protein molecule (n) was 0.8 (CHOL), 0.7 (DOPE), 1.0 (DDAB), and 1.3 (DOTAP). Molecular modeling showed the participation of several amino acid residues in lipid-protein complexation with the order of binding DOTAP > DDAB > DOPE > CHOL. Alterations of the protein conformation were observed in the presence of lipids with a minor decrease in beta-sheet and an increase in turn structure.