Jean Dufourcq

Bending elasticities of model membranes: influences of temperature and sterol content.

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.

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.

Conformational change and self association of monomeric melittin

Melittin is a well-known amphipathic peptide which acts as a direct lytic factor on biological membranes [ 11. Its ability to induce leakage both on natural membranes and liposomes has been clearly demonstrated [2]. Melittin-phospholipid interactions have also been extensively studied [3-51, however few studies have been dealing with the structure of the peptide itself, in solution. It is known to behave as a tetramer [ 1,6]; it is also assumed from an early circular dichroism study [7] to be mainly in random coil. The possibility of conformational and aggregation changes induced by organic solvent or phospholipids was pointed out [8,9] but no definite experimental proof was presented on these problems. Here, we report the existence of melittin as a monomer and give the parameters which govern the self-association of the peptide. We also present arguments indicating that the polymerization process involves important changes in the secondary structure of melittin. These findings allow a more coherent picture of the behaviour of the peptide in solution.

Intrinsic fluorescence study of lipid-protein interactions in membrane models. Binding of melittin, an amphipathic peptide, to phospholipid vesicles

Abstract Melittin is a small peptide extracted from bee venom, which has a direct lytic activity on living cells, and equally disrupts the liposome structure. In agreement with a previous work of Mollay, C. and Kreil, G. ((1973) Biochim. Biophys. Acta 316, 196–203), it is shown that intrinsic fluorescence of the only tryptophan of melittin is very sensitive to the binding to phospholipids. The observed blue shifts, from 352 to 333 nm, upon addition of lipid vesicles, indicate that the Trp residue is going from a polar to a non-polar environment, and clearly show that melittin displays hydrophobic interaction with zwitter-ionic or negative phospholipids, whatever the ionic strength or pH. The hydrophobic nature of the interactions is confirmed by the sensitivity of the fluorescence intensity of the Trp residue to the phase transitions of phosphatidylserine, dimyristoyl and dipalmitoyl phosphatidylcholine, which implies a close contact between this residue and aliphatic chains. It is also shown that the length of the aliphatic chains has no significant effect on binding, but that their fluidity is a critical parameter. Binding is indeed much less efficient when aliphatic chains are in their crystalline state, below the phase transition temperature. Binding is strongly dependent on the net electrical charge borne by the vesicles. The effect of a pH increase, or of an addition of dicetylphosphate to phosphatidylcholine vesicles leads to the conclusion that binding is enhanced by an increase of the net negative charge of the lipid bilayer. This result is illustrated by the fact that melittin is totally bound when the lipid to protein molar ratio is equal to 3 or 4 in the case of phosphatidylserine, and about 25 for phosphatidylcholines. Phosphatidylserine vesicles can then bind up to 8 times more melittin than do phosphatidylcholines. In conclusion, both electrostatic and hydrophobic forces have to be considered as important binding parameters: the first step could be an ionic interaction between Lys and Arg residues of melittin and negative groups of phospholipids, either phosphate or carboxylic, the second step being the insertion of hydrophobic residues within the bilayer, this involving at least the Trp residue, but probably all the hydrophobic part of the peptide.

Investigations at the air/water interface using polarization modulation IR spectroscopy

The ability of polarization modulation IR reflection absorption spectroscopy (PM–IRRAS) to study the air/water interface is presented. A brief description of the set-up and of the experimental procedure is given. Theoretical simulations accounting for the uniaxial nature of the spread monolayer lead to optimum experimental conditions (71 ° for the angle of incidence) and to a specific surface selection rule. Application to the study of cadmium arachidate, dimyristolyl phosphatidylcholine (DMPC) and polypeptidic Langmuir films illustrates the potential uses of this method.

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.

The self-association of melittin and its binding to lipids: an intrinsic fluorescence polarization study.

Intrinsic fluorescence has proved to be very useful for studying protein-protein or lipid-protein interactions [l--8 J. Although these studies are mostly restricted to the analysis of intensity or wavelength changes in the emission spectra, polarization measurements can afford valuable information, as shown in the self-association of apo-lipoproteins [7,8] and the interaction of glucagon with lipids [5,6]. Here, intrinsic fluorescence polarization is applied to the study of the self-association of mellitin, and of its binding to lipids. Melittin is a small amphipathic peptide of 26 residues, extracted from bee venom, which is known to have a direct lytic activity on living cells [9-l 1 J. It contains only one fluorescent residue, Trp,,, and its emission spectrum is very sensitive to interactions with lipids [I ,3].

Structure, orientation and affinity for interfaces and lipids of ideally amphipathic lytic LiKj(i=2j) peptides

The behavior of lytic ideally amphipathic peptides of generic composition LiKj(i=2j) and named LKn, n=i+j, is investigated in situ by the monolayer technique combined with the recently developed polarization modulation IR spectroscopy (PMIRRAS). A change in the secondary structure occurs versus peptide length. Peptides longer than 12 residues fold into alpha-helices at interfaces as expected from their design, while enough shorter peptides, from 9 down to 5 residues, form intermolecular antiparallel beta-sheets. Analysis of experimental and calculated PMIRRAS spectra in the amide I and II regions show that peptides are flat oriented at the interfaces. Structures and orientation are preserved whatever the nature of the interface, air/water or DMPC monolayer, and the lateral pressure. Peptide partition constants, KaffPi, are estimated from isobar surface increases of DMPC monolayers. They strongly increase when Pi decreases from 30 mN/m to 8 mN/m and they vary with peptide length with an optimum for 12 residues. This non-monotonous dependence fits with data obtained in bilayers and follows the hemolytic activity of the peptides. Lipid perturbations due to peptide insertion essentially detected on the PO4- and CO bands indicate disorder of the lipid head groups. Lysis induced on membranes by such peptides is proposed to first result from their flat asymmetric insertion.

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.

Ideally amphipathic β-sheeted peptides at interfaces: structure, orientation, affinities for lipids and hemolytic activity of (KL)mK peptides

Abstract Designed to model ideally amphipathic β-sheets, the minimalist linear (KL)mK peptides (m=4–7) were synthesized and proved to form stable films at the air/water interface, they insert into compressed dimyristoylphosphatidylcholine monolayers and interact with egg phosphatidylcholine vesicles. Whatever the interface or the lateral pressure applied to the films, FT-IR and polarization-modulated IRRAS spectroscopy developed in situ on the films indicated that all the peptides totally fold into intermolecular antiparallel β-sheets. Calculated spectra of the amide region allowed us to define the orientation of the β-strands compared to the interface. It is concluded that such β-sheets remain flat-oriented without deep perturbation of zwitterionic phospholipids. Dansyl labelling at the N-terminus indicates that all the peptides are monomeric at a low concentration in aqueous buffer and bind to lipids with similar Dns burying. The affinities for zwitterionic lecithin mono- and bilayers, quantitatively estimated from buffer to lipid partition constants, monotonically increased with peptide length, indicating that hydrophobicity is a limiting parameter for lipid and membrane affinities. Peptides induced permeability increases on zwitterionic liposomes, they are strongly hemolytic towards human erythrocytes and their activity increases concurrently with length. Taking into account the lipid affinity, a hemolytic efficiency can be defined: at the same amount of peptide bound, this efficiency strongly increases with the peptide length. It is proposed that the first determinant step of membrane disturbance is the invasion of the outer membrane leaflet by these ideally amphipathic β-sheeted structures lying flat at the interface, like large rafts depending on the number of β-strands.