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Free Content Insertion of Escherichia coliα-haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment

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α-Haemolysin is an extracellular protein toxin (≈107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified α-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze–fracture electron microscopy. All the data concur in indicating that α-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted α-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic α-helices, appropriate for protein–lipid interaction, but no hydrophobic transmembrane helices are predicted in α-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by α-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of α-haemolysin as a pore-forming toxin.
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Document Type: Research Article

Affiliations: 1: Grupo Biomembranas (Unidad Asociada al C.S.I.C.), Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain., 2: Instituto Cajal, C.S.I.C., Madrid, Spain., 3: Faculté Universitaire des Sciences Agronomiques, Gembloux, Belgium.

Publication date: February 1, 1999

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