Jean-François Faucon

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.

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.

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].

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Abstract Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C 14 lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C 16 lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C 16 aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C 14 chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.

Aliphatic chain transitions of phospholipid vesicles and phospholipid dispersions determined by polarization of fluoroscence

Abstract The polarization of fluorescence of dansyl phosphatidylethanolamine and 9-methylanthracene shows that these compounds are reliable indicators of the order-disorder transitions of the phospholipid aliphatic chains in bilayer systems. The transition is better defined in phospholipid dispersion than in vesicles. It is concluded that the two models are not identical as far as the structure near the melting temperature is concerned. Experiments in turbid solutions were performed with horizontal slits either in the incident or emitted beams, which eliminate the effect of light scattering. This improvement in experimental technique may facilitate the fluorescence polarization study of membrane suspensions.

Study of lipid—protein interactions in membrane models: Intrinsic fluorescence of cytochrome b5—phospholipid complexes

Until now, few integral membrane proteins have been isolated and studied; cytochrome h5 is one of them. Strittmatter et al. [I ] and Ememoto and Sato [2] first demonstrated that pure detergent extracted cytochrome h5 can bind to microsomes. Furthermore, Sullivan and Holloway [3] showed that this protein interacts with egg phosphatidylcholine. By reconstitution experiments with single shell vesicles? Dufourcq et al. [4] and Robinson and Tanford [S] obtained a more detailed knowledge of the lipid-protein complexes. Cytochrome b5 can be described as a protein composed of two different moieties: one. bearing the heme, is hydrophilic and can be obtained by trypsin action on microsomes (cytochrome t-b,). The second one is composed of about fifty four residues for bovine liver extracted protein, and would be responsible for the binding of the whole protein to the membrane [6,7]. Furthermore, this hydrophobic peptide contains several aromatic residues; four of them are tryptophan residues [8]. Fluorescence is a classical method to study the environment of aromatic residues in soluble proteins [9,10]. This technique has also been used to look at interactions between apolipoproteins and lipids [11,12,13]. In this paper, we report variations of the fluorescence parameters during interactions of purified bovine liver cytochrome b5 with selected phospholipids, in order to obtain information on the structure of the lipidprotein complexes as an approach to the structure of membranes. 2. Materials and methods

Acyl chain length dependence in the stability of melittin-phosphatidylcholine complexes. A light scattering and 31P-NMR study

Light scattering and 31P-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C16:0 to C20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, Ri > or = 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 A with dipalmitoyl-PC (DPPC) to 1500 A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 A radius when the system is cooled down below Tc (the gel-to-fluid phase transition temperature). Little chain length dependence is observed for the small objects. Small structures are also detected independently of the physical state of lipids (gel or fluid) when Ri < or = 5 and provided the interaction has been made above Tc. Small discs clearly characterized for DPPC and distearoyl-PC (DSPC) lipids are much less stable with DAPC. However in the long term, all these small structures fuse into large lipid lamellae. Discs are thermodynamically unstable and kinetics of disappearance of the small lipid-toxin complexes increases as the chain length increases in the sense: DAPC >> DSPC > DPPC. Kinetics of fusion of the small discs into extended bilayers is described by a pseudo-first-order law involving a lag time after which fusion starts. Increasing the chain length decreases the lag time and increases the rate of fusion. Formation of both the large vesicles in the fluid phase and the small discs in the gel phase as well as their stability is discussed in terms of relative shapes and dynamics of both lipids and toxin.

Perturbation of binary phospholipid mixtures by melittin: A fluorescence and Raman spectroscopy study

The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.

Peptide-membrane interactions A fluorescence study of the binding of oligopeptides containing aromatic and basic residues to phospholipid vesicles

Abstract The binding of oligopeptides containing basic and aromatic residues to phospholipid vesicles has been studied by fluorescence spectroscopy. Tryptophan-containing peptide such as Lys-Trp-Lys or Lys-Trp(OMe) exhibit a shift of their fluorescence toward shorter wavelengths and an increased fluorescence quantum yield upon binding to phosphatidylinositol (PI) or phosphatidylserine (PS) vesicles. No binding was detected with phosphatidylcholine vesicles. The binding is strongly dependent on ionic strength and pH. Binding decreases when ionic strength increases indicating an important role of electrostatic interactions. The pH-dependence of binding reveals that the apparent p K of the terminal carboxyl group of Lys-Trp-Lys is raised by ∼3 units upon binding to PI and PS vesicles. The binding of tyrosine-containing peptides to PI and PS vesicles is characterized by an increase in the fluorescence quantum yield of the peptide without any shift in fluorescence maximum. A natural nonapeptide from the myelin basic protein which contains one tryptophan residue binds to PI and PS vesicles at low pH when the acidic groups are neutralized. This binding is accompanied by a shift of the tryptophyl fluorescence toward shorter wavelengths together with an enhancement of the fluorescence quantum yield. Dissociation of the complex is achieved at high ionic strength. These results indicate that aromatic residues of oligopeptides bound to the phospholipid polar heads by electrostatic interactions become buried in a more hydrophobic environment in the vicinity of the aliphatic chains of the lipids.

Lipid-protein interactions in membrane models. Fluorescence polarization study of cytochrome b5-phospholipids complexes.

According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5. Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very sensitive to the binding of proteins, and so is a useful method to study lipid-protein interactions. The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of bilayer, only due to the interaction of the hydrophobic peptide tail. Interaction of dipalmitoly phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 degrees C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected. Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.