Location of tryptophan residues in free and membrane bound Escherichia coli alpha-hemolysin and their role on the lytic membrane properties.
alpha-hemolysin (HlyA) is an extracellular protein toxin secreted by Escherichia coli that acts at the level of plasma cell membranes of target eukaryotic cells. Previous studies showed that toxin binding to the bilayers occurs in at least two ways, a reversible adsorption and an irreversible insertion. Studies of HlyA insertion into bilayers formed from phosphatidylcholine show that insertion is accompanied by an increase in the protein intrinsic fluorescence. In order to better define structural parameters of the membrane-bound form, the location of tryptophan residues was studied by means of quenchers of their intrinsic fluorescence located at 7, 12 and 16 positions of the acyl chain of phosphatidylcholine. The quenching was progressively weaker suggesting an interfacial location of the Trp. In parallel, HlyA was subjected to oxidation with N-bromosuccinimide to study the role of Trp residues exposed to aqueous media in its structure-function relationship. In the folded toxin molecule, a single residue was susceptible to oxidation with NBS, whereas incubation with LUV of the toxin prior modification prevents its oxidation, suggesting that Trp residue(s) are directly involved in toxin binding and insertion. Finally, the modification of residues exposed to solvent resulted in a complete impairment of the lytic activity. It was concluded that the modification-sensitive Trp residues are essential for the structure and function of native HlyA. These results are consistent with the model proposed by Soloaga et al. (Mol. Microbiol. 31 (1999) 1013-1024) according to which HlyA is bound to a single monolayer through a number of amphipathic instead of inserted transmembrane helices.
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Instituto de Investigaciones Bioquímicas (INIBIOLP), Facultad de Ciencias Médicas, CONICET, 60 y 120 (1900), La Plata, Argentina.
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D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D001974:Bromosuccinimide; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers; D014364:Tryptophan; D001974:Bromosuccinimide