Interaction mode specific reorganization of gel phase monoglyceride bilayers by beta-lactoglobulin

The interaction between beta-lactoglobulin and sonicated aqueous dispersion s of the gel phase forming monoglyceride monostearoylglycerol were studied using isothermal titration calorimetry, direct binding experiments, differe ntial scanning calorimetry, leakage of a fluorescent dye and solid-state P- 31- and H-2-NMR. In the absence of a charged amphiphile, monostearoylglycer ol forms a precipitate. Under these conditions, no interaction with beta-la ctoglobulin was observed. In the presence of the negatively charged amphiph ile dicetylphosphate, the gel phase monostearoylglycerol formed stable and closed, probably unilamellar, vesicles with an average diameter of 465 nm. beta-Lactoglobulin interacts with these bilayer structures at pH 4, where t he protein is positively charged, as well as at pH 7 where the protein is n egatively charged. Under both conditions of pH, the binding affinity of bet a-lactoglobulin is in the micromolar range as observed with ITC and the dir ect binding assay. At pH 4, two binding modes were found, one of which is d etermined with ITC while the direct binding assay determines the net result of both. The first binding mode is observed with ITC and is characterized by a large binding enthalpy, a decreased enthalpy of the MSG L-beta to L-al pha phase transition and leakage of a fluorescent dye. These characteristic s are explained by a beta-lactoglobulin induced partial L-beta to coagel ph ase transition that results from a specific electrostatic interaction betwe en the protein and the charged amphiphile. This explanation is confirmed by solid-state H-2-NMR using 1-monostearoylglycerol with a fully deuterated a cyl chain. Upon interaction with beta-lactoglobulin, the isotropic signal i n the H-2-NMR spectrum of the monostearoylglycerol-dicetylphosphate mixture partially transforms into a broad anisotropic signal which could be assign ed to coagel formation. The second binding mode probably results from an as pecific electrostatic attraction between the negatively charged bilayer and the positively charged protein and causes the precipitation of the dispers ion. At pH 7, only the first binding mode is observed. (C) 1999 Elsevier Sc ience B.V. All rights reserved.