Thermotropic and dynamic characterization of interactions of acylated α-bungarotoxin with phospholipid bilayer membranes

Bruce Babbitt, Leaf Huang, Ernesto I Freire

Research output: Contribution to journalArticle

Abstract

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 (Tm), 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 Tm of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below Tm while increasing the order above Tm. 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 Tm the rotational mobility is only slightly restricted. Lateral diffusion measurements of fluorescein-labeled PBGT (PBGTF) associated with multilamellar liposomes composed of egg PC or DPPC measured by fluorescence recovery after photobleaching technique indicate that the lateral mobility of PBGTF is dependent upon the bilayer fluidity. Lateral diffusion coefficients at 25°C of (1.7 ± 1.2) × 10-8 cm2/s (egg PC) and 1 × 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.

Original languageEnglish (US)
Pages (from-to)3920-3926
Number of pages7
JournalBiochemistry®
Volume23
Issue number17
StatePublished - 1984

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ASJC Scopus subject areas

  • Biochemistry

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