Abstract
alpha-Haemolysin is an extracellular protein toxin (107KDa) produced by certain pathogenic strains of Escherichia coli. It can bind to lipid bilayers and produce membrane disruption in cell and model membranes. Protein secondary structure predictions suggest an amphipathic helix conformation for the membrane-interacting domain of HlyA, and no potential transmembrane segments. In this paper, we investigated the lipid associating properties of the chemically synthesized peptide H9 (MFEHVASKMADVIAEWEK) corresponding to residues 417-434 of HlyA. Changes in the intrinsic fluorescence and fluorescence anisotropy of the single Trp residue after the addition of DMPC-LUV reveal that the peptide-membrane interaction is optimum at or above the gel-liquid crystalline transition temperature of the lipid. Moreover, the peptide induces vesicle aggregation, as detected through changes in light scattering by the vesicle suspension. However, under comparable conditions, H9 has no hemolytic activity against sheep eryth rocytes, nor does it induce leakage of vesicular aqueous contents. These data suggest that HlyA binds the membrane surface through amphipathic helices, though cooperativity among neighbouring helices is probably necessary to explain the lytic effect of the toxin.
Keywords: AMPHIPATHIC PEPTIDE, ESCHERICHIA coli HAEMOLYSIN, a-Haemolysin, Intrinsic fluorescence measurements, Steady state fluorescence anisotropy, Merocyanine 540 absorption spectra, Haemolytic assays
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Frontiers in Protein and Peptide Sciences
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