Insertion of Escherichia coli alpha-haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment

alpha-Haemolysin is an extracellular protein toxin (approximate to 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membrane s of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that rec eptor-mediated binding is not essential, In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylchol ine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer syst ems containing bound protein have been examined by differential scanning ca lorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in ind icating that alpha-haemolysin, under conditions leading to cell lysis, beco mes inserted in the target membrane in the way of intrinsic or integral pro teins. In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers , i.e. it is not a transmembrane protein. The experimental data are complem ented by structure prediction studies according to which as many as ten amp hipathic alpha-helices, appropriate for protein-lipid interaction, but no h ydrophobic transmembrane helices are predicted in alpha-haemolysin. These o bservations and predictions have important consequences for the mechanism o f cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrang ement of the toxin in the target membrane is not compatible with the concep t of alpha-haemolysin as a pore-forming toxin.