Thermotropic and Dynamic Characterization of Interactions of Acylated α-Bungarotoxin with Phospholipid Bilayer Membranes

The interactions of palmitoyl-α-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (T_m), a broadening of the heat capacity function, and a decrease in the enthalpy change associated with the phospholipid gel to liquid-crystalline transition. Analysis of the dependence of this decreased enthalpy change on the protein/lipid molar ratio indicates that each PBGT molecule exhibits a localized effect upon the bilayer, preventing approximately six lipid molecules from participating in the lipid phase transition. Additional calorimetric experiments indicate that binding to acetylcholine receptor enriched membranes causes a small increase in the T_m of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-di-phenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below T_m while increasing the order above T_m. These results have been further supported by rotational mobility measurements of erythrosin-labeled PBGT associated with giant (about 2-μm) unilamellar vesicles composed of dielaidoylphosphatidylcholine or dioleoylphosphatidylcholine using the time-dependent decay of delayed fluorescence/phosphorescence emission anisotropy. Rotational correlation times in the submillisecond time scale (about 30 μs) indicate that the protein is highly mobile in the fluid phase and that below T_m the rotational mobility is only slightly restricted. Lateral diffusion measurements of fluorescein-la-beled PBGT (PBGT^F) associated with multilamellar liposomes composed of egg PC or DPPC measured by fluorescence recovery after photobleaching technique indicate that the lateral mobility of PBGT^F is dependent upon the bilayer fluidity. Lateral diffusion coefficients at 25 °C of (1.7 ± 1.2) X 10^-8 cm²/s (egg PC) and 1 X 10^-11 cm²/s (DPPC) were obtained. These results are consistent with a model in which PBGT interacts with the phospholipid bilayer principally via the fatty acid chain while the polypeptide region of the molecule does not interact strongly with the bilayer.