Jean-Louis Dasseux

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.

The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin.

An original serie of 12‐ to 22‐residue‐long peptides was developed, they are only constituted by apolar Leu and charged Lys residues periodically located in the sequence in order to generate ideal highly amphipathic α‐helices. By circular dichroism, the peptides are proven to be mainly α‐helical in organic and aqueous solvents and in the presence of lipids. The peptides are highly hemolytic, their activity varies according to the peptide length. The 15‐, 20‐, and 22‐residue‐long‐peptides have LD50 ∼5 × 10−8 M for 107 erythrocytes, i.e. they are 5–10 times more active than melittin, and are indeed several orders of magnitude more active than magainin or mastoparan.

Lipid-Protein Interactions: A Reinvestigation of Melittin Induced Effects on the Structure and Dynamics of Phosphatidylcholines

Perturbations induced by melittin (Mel) on the thermotropism of synthetic lecithins, respectively,dimyristoyl-, dipal-mitoyl- and distearoyl-phosphatidylcholine (DMPC, DPPC, DSPC) are investigated and rationalized from data obtained by fluorescence polarization (P), differential scanning calorimetry (DSC), Raman spectroscopy, and freeze-fracture electron microscopy.