title: Interaction mode specific reorganization of gel phase monoglyceride bilayers by beta-lactoglobulin.
abstract: The interaction between beta-lactoglobulin and sonicated aqueous dispersions of the gel phase forming monoglyceride monostearoylglycerol were studied using isothermal titration calorimetry, direct binding experiments, differential scanning calorimetry, leakage of a fluorescent dye and solid-state (31)P- and (2)H-NMR. In the absence of a charged amphiphile, monostearoylglycerol forms a precipitate. Under these conditions, no interaction with beta-lactoglobulin was observed. In the presence of the negatively charged amphiphile 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 the protein is positively charged, as well as at pH 7 where the protein is negatively charged. Under both conditions of pH, the binding affinity of beta-lactoglobulin is in the micromolar range as observed with ITC and the direct binding assay. At pH 4, two binding modes were found, one of which is determined 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(alpha) phase transition and leakage of a fluorescent dye. These characteristics are explained by a beta-lactoglobulin induced partial L(beta) to coagel phase transition that results from a specific electrostatic interaction between the protein and the charged amphiphile. This explanation is confirmed by solid-state (2)H-NMR using 1-monostearoylglycerol with a fully deuterated acyl chain. Upon interaction with beta-lactoglobulin, the isotropic signal in the (2)H-NMR spectrum of the monostearoylglycerol-dicetylphosphate mixture partially transforms into a broad anisotropic signal which could be assigned to coagel formation. The second binding mode probably results from an aspecific electrostatic attraction between the negatively charged bilayer and the positively charged protein and causes the precipitation of the dispersion. At pH 7, only the first binding mode is observed.
authors.affiliation: Department Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, Netherlands.
authors.forename: J W
authors.initials: JW
authors.lastname: Boots
authors.forename: V
authors.initials: V
authors.lastname: Chupin
authors.forename: J A
authors.initials: JA
authors.lastname: Killian
authors.forename: R A
authors.initials: RA
authors.lastname: Demel
authors.forename: B
authors.initials: B
authors.lastname: de Kruijff
mesh_terms: D000818:Animals; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D005456:Fluorescent Dyes; D005782:Gels; D005989:Glycerides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007782:Lactoglobulins / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D055672:Static Electricity; D014966:Xanthenes
chemical_list: D005456:Fluorescent Dyes; D005782:Gels; D005989:Glycerides; D007782:Lactoglobulins; D008051:Lipid Bilayers; D014966:Xanthenes; C034657:Texas red