Does alpha hemolysin of E. coli form an oligomer structure when it is bound to membranes?

V. HERLAX; L. BAKÁS

Pinamar,Argentina

Congreso; X PABMB congress, XLI Reunión anual SAIB, XX Reunión anual SAN; 2005

PABMB, SAIB y SAN

  Alpha hemolysin is an extracellular protein toxin (107 kDa) secreted by Escherichia coli that acts at the level of plasma membranes of target eukaryotic cells. Postraslational modification of the protein with fatty acids is required for all known cytotoxic activities which occurs at two internal lysine residues (K564 y K690).   This toxin promotes the formation of proteo-lipidic pores at lytic concentration rather than forming purely proteinaceous ionic channels.   We studied the interaction of this toxin with phospholipid membrane using artificial planar lipid membranes composed of asolectin. Addition of nanomolar concentrations of toxin resulted in an increase of bilayer conductance at a step concentration dependent fashion, suggesting that several toxin molecules could be involved in the conductive unit. To obtain conclusive information on the formation of the oligomer we used different cystein mutants of the toxin. These mutants were derivatized with fluorescent probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives) in order to study the formation of an oligomer on red blood cell membranes by Fluorescent Resonance Energy Transfer (FRET). It seems that the acyl chains may be involved in the oligomerization process.