Contribution of individual tryptophan residues to the spectroscopic and functional properties of Escherichia coli ƒÑ-hemolysin¡¨

V. HERLAX; L. BAKÁS

San Pablo

Congreso; International Workshop on Spectroscopy for Biology; 2001

   Alpha hemolysin (HlyA) is an extracelular protein virulence factor found in certain urophathogenic strains of E. coli.It is Knowm to discrupt eukaryotic cell membranes altering celullar activity and lysing them.Aditional details about lipid-protein interactions can be obtain fron spectoscopic studies of the intrinsic fluorescense. HlyA has 4 tryptophans (W) which can serve as intrinsic flurophores. They are located at positions 431, 480, 579 y 914.Measurement of fluorescence spectra of Hlya ant its mutants in which one W residue has been replaced by a leucine (L) to evaluate the individual W contribution. These results indicate that the mutation did not have long range effects on the protein structure. The comparison of fluorescense spectra of wild type and mutant protein indicates that the net enrgy transfer process takes place among different W residues. In parallel HlyA was subjected  to oxidation with N-bromosuccinimide to study the exposure of the W residues to the aqueous media.In order to better define the role of individual W in the protein-membrane interaction we studied the fluorescence energy transfer (FRET) from W to laurdan incorporeted into membranes.These results indicate that there are no lytic residue critical for the lytic activity, though W 579 appears to be essential to the cytotoxic effect, indicating that both effects (lytic and cytotoxic) the interacting protein-lipid domains are differents. Yet toxin-receptor interaction migth be involved in this cytotoxic effect.