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Biophysical Journal 84:2382-2392 (2003)
© 2003 The Biophysical Society

Effects of the Eukaryotic Pore-Forming Cytolysin Equinatoxin II on Lipid Membranes and the Role of Sphingomyelin

Boyan B. Bonev*, Yuen-Han Lam{dagger}, Gregor Anderluh{ddagger}, Anthony Watts*, Raymond S. Norton§ and Frances Separovic{dagger}

* Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; {dagger} School of Chemistry, University of Melbourne, Victoria 3010, Australia; {ddagger} Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia; and § Biomolecular Research Institute, 343 Royal Parade, Parkville 3052, Australia

Correspondence: Address reprint requests to Frances Separovic, School of Chemistry, University of Melbourne, VIC 3010, Australia. Tel.: 61-3-8344-6464; Fax: 61-3-9347-5180; E-mail: fs{at}unimelb.edu.au.

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by 31P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20–40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state 31P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The 2H and 31P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.




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