Odile Siffert

Interaction of staphylococcal δ‐toxin and synthetic analogues with erythrocytes and phospholipid vesicles

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.

Dynamics and orientation of amphipathic peptides in solution and bound to membranes: a steady-state and time-resolved fluorescence study of staphylococcal δ-toxin and its synthetic analogues

Abstract. The environment of both the hydrophilic and hydrophobic sides of α-helical δ-toxin are probed by tryptophanyl (Trp) fluorescence, when self-association occurs in solution and on binding to membranes. The fluorescence parameters of staphylococcal δ-toxin (Trp15 on the polar side of the amphipathic helix) and synthetic analogues with single Trp at position 5 or 16 (on the apolar side) were studied. The time-resolved fluorescence decays of the peptides in solution show that the local environment of their single Trp is always heterogeneous. Although the self-association degree increases with concentration, as shown by fluorescence anisotropy decays, the lifetimes (and their statistical weight) of Trp16 do not change, contrary to what is observed for Trp15. The first step of self-association is then driven by hydrophobic interactions between apolar sides of α-helices, whilst further oligomerization involves their polar side (Trp15) via electrostatic interactions. This is supported by dissociation induced by salt. For all self-associated peptides, the polarity of the Trp microenvironment was not significantly modified upon binding to phospholipid vesicles, as indicated by the small shifts of the fluorescence emission spectra and lifetime values. However, the relative populations of the lifetime classes vary with bound-peptide density similar to the rates of their global motions in bilayers or smaller particles. Quenching experiments by water or lipid-soluble compounds show changes of the orientation of membrane-inserted peptides, from probably dimers lying flat at the interface at low peptide density, to oligomers spanning the membrane and inducing membrane fragmentation at high peptide density.

Optical measurement of a solvent-induced isomerization in a proline-containing hexapeptide

Abstract The hexapeptide Gly-Gly-Pro-Tyr-Gly-Gly has been synthesized and its tyrosine residue converted to nitrotyrosine by reaction with tetranitromethane. When diluted from dimethylsulfoxide into aqueous solution, the nitrated hexapeptide undergoes a slow conformational change characterized by a change in the ionization state of the nitrotyrosine group. This slow reaction is not observed with peptides containing nitrotyrosine and no proline. Also, the rate and activation enthalpy of this slow conformational change suggest that it could be due to proline cis-trans isomerization. The possibility of measuring the rate of cis-trans isomerization of proline residues in a polypeptide chain is discussed.

Characteristics of guinea-pig immune sera elicited by a synthetic diphtheria toxin oligopeptide.

Several oligopeptides of different lengths contained within the Cys 186-Cys 201 first disulfide loop of the diphtheria toxin molecule have been synthesized by a solid-phase method. 125I-labeled rabbit antibodies raised against diphtheria toxin reacted specifically with oligopeptides linked to m-nitrobenzhydrylamine resin when the amino acid chain length was equal to or greater than 10 residues. The synthetic tetradecapeptide (STDP) corresponding to the sequence Gly 188-Cys 201 was used to immunize guinea-pigs. The immune sera obtained reacted with the whole diphtheria toxin molecule as judged by an antigen-linked immunosorbent assay. Anti-STDP sera exhibited a clear, albeit limited, neutralizing effect against the lethal action of diphtheria toxin on cultivated Vero cells. The anti-STDP sera were also able to partially block the ADP-ribosylation of elongation factor 2 mediated by whole diphtheria toxin. In contrast, anti-STDP sera were almost inactive on the enzymic activities of either toxin fragment A or crm 45, a mutant protein which lacks the 15,000 mol. wt C-terminal sequence of the toxin molecule. On the basis of the results obtained, a possible localization of the Cys 188-Cys 201 loop region on the toxin molecule is proposed.