Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence, monolayer and modeling.

A cationic amphiphilic peptide made of 10 leucine and 10 lysine residues, and four of its fluorescent derivatives in which leucines were substituted by Trp residues at different locations on the primary sequence have been synthesized. The interactions of these five peptides with neutral anionic or cationic vesicles were investigated using circular dichroism, steady state and time-resolved fluorescence with a combination of Trp quenching by brominated lipid probes, monolayers, modeling with minimization and simulated annealing procedures. We show that all the five peptides interact with neutral and anionic DMPC, DMPG, DOPC or egg yolk PC vesicles. The binding takes place whatever the peptide conformation in solution is. In the case of DMPC bilayers the binding free energy DeltaG is estimated at -8 kcal mole-1 and the number of phospholipid molecules involved is about 20-25 per peptide molecule. Peptides are bound as single-stranded alpha helices orientated parallel to the bilayer surface. In the anchoring of phospholipid head groups around the peptides, the lipid molecules are not smeared out in a plane parallel to the membrane surface but are organized around the hydrophilic face of the alpha helices like 'wheat grains around an ear' and protrude outside the bilayer towards the solvent. We suggest that such a lipid arrangement generates transient structural defects responsible for the membrane permeability enhancement. When an electrical potential is applied, the axis of the peptide helices remains parallel to the membrane surface and does not reorient to give rise to a bundle of helix monomers that forms transmembrane channels via a 'barrel stave' mechanism. The penetration depth of alpha helices in relation to the position of phosphorus atoms in the unperturbed lipid leaflet is estimated at 3.2 A.

Electric field-induced fusion of mitochondria.

Fusion of mitochondria in H-medium from rat liver was induced by the application of square-wave voltage with electric field strengths of 1-2.5 kV/cm and duration 100 microseconds. Electron micrographs showed that the newly fused mitochondria could contain up to five mitoplasts. The fusion yield was close to 12% and respiratory activity was enhanced. The electric field lines did not go through the inner membrane, however, when the electric field strength was greater than 3 kV/cm they did so, resulting in total destruction of the mitochondria.

Interactions of basic amphiphilic peptides with dimyristoylphosphatidylcholine small unilamellar vesicles: optical, NMR, and electron microscopy studies and conformational calculations.

The interactions of DMPC small unilamellar vesicles with four amphiphilic polypeptides [(LKKL)n, (LRRL)n, (LKKL)4, and (YKKY)n] have been investigated by circular and infrared dichroism, turbidimetry, electron microscopy, and fluorescence, 1H, and 31P nuclear magnetic resonance spectroscopy. The main results obtained are the following: (1) Well-defined complexes are formed by the association of one amino acid residue with approximately two lipid molecules. (2) In the presence of polypeptides fusions are observed between SUVs when the molar ratio p is less than 0.05, and a clearance effect is observed when p is higher than 0.05. (3) The anchoring sites of the polypeptides on DMPC molecules are the negative phosphate groups through electrostatic interactions with the terminal NH3+ of lysine residues. (4) The polypeptides adopt an alpha-helical conformation with their axis parallel to the membrane surface. The hydrophobic part of the amphiphilic alpha helix can penetrate the outer lipid leaflet down to the C5 position. (5) Choline methyl groups are not involved in the interactions between lipid molecules and amino acid residues. (6) Phosphorus atom mobility around the P-O-glycerol bond is strongly reduced whereas that of methylene groups is progressively weakened when going up from C13 to C1. Finally, using modeling and energy calculations a model of possible Ac(LKKL)4NHEt-DMPC SUV complexes is presented.