Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s(o)) and liquid-disordered (l(d)) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l(o)) and l(d) phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s(o)-l(d) and l(o)-l(d) phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Melittin-induced leakage from phosphatidylcholine vesicles is modulated by cholesterol: a property used for membrane targeting

Melittin, an amphiphathic peptide, affects the permeability of vesicles. This can be demonstrated using the dye release technique. Calcein, a fluorescent marker, is trapped in large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) vesicles and melittin-induced leakage of the dye can be monitored directly by increasing fluorescence intensity. First, we characterized the effect of increasing cholesterol content in the membrane on melittin-induced leakage and our results reveal that cholesterol inhibits the lytic activity of the peptide. Using intrinsic fluorescence of the single tryptophan of melittin and ²H-NMR of headgroup deuterated phosphatidylcholine, we demonstrated that the affinity of melittin for phosphatidylcholine vesicles is reduced in the presence of cholesterol; this is associated with the tighter lipid packing of the cholesterol-containing bilayer. This reduced binding is responsible for the reduced melittin-induced leakage from cholesterol-containing membranes. The pathway of release was determined to be an all-or-none mechanism. Finally, we investigated the possibility of achieving specific membrane targeting with melittin, when vesicles of different lipid composition are simultaneously present. Melittin incubated together with vesicles made of pure POPC and POPC containing 30(mol)% cholesterol can empty nearly all the cholesterol-free vesicles while the cholesterol-containing vesicles remain almost intact. Owing to the preferential interaction of melittin with the pure POPC vesicles, we were able to achieve controlled release of encapsulated material from a specific vesicle population. Received: 8 May 1996 / Accepted: 12 September 1996     

Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).     

Effect of phospholipid composition on an amphipathic peptide-mediated pore formation in bilayer vesicles

To better understand the influence of phospholipid acyl-chain composition on the formation of pores by cytotoxic amphipathic helices in biological membranes, the leakage of aqueous contents induced by the synthetic peptide GALA (WEAALAEALAE ALAEHLAEALAEALEALAA) from large unilamellar phospholipid vesicles of various compositions has been studied. Peptide-mediated leakage was examined at pH 5.0 from vesicles made of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with the following acyl-chain compositions: 1-palmitoyl-2-oleoyl (PO), 1,2-dioleoyl (DO), 1, 2-dielaidoyl (DE), and 1,2-dipetroselinoyl (DPe). A mathematical model predicts and simulates the final extents of GALA-mediated leakage of 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) from 1-palmitoyl-2-oleoyl-phosphatidylcholine/1-palmitoyl-2-oleoyl-phospha tidylglycerol (POPC/POPG) and 1, 2-dielaidoyl-sn-glycero-3-phosphocholine/1, 2-dielaidoyl-phosphatidylglycerol (DEPC/DEPG) liposomes at pH 5.0 as a function of peptide concentration in the bilayer, by considering that GALA pores responsible for this leakage have a minimum size of 10 +/- 2 monomers and are formed by quasiirreversible aggregation of the peptide. With the phospholipid acyl-chain compositions tested, GALA-induced ANTS/DPX leakage follows the rank order POPC/POPG approximately DEPC/DEPG > DPePC/DPePG > DOPC/DOPG. Results from binding experiments reveal that this reduced leakage from DOPC/DOPG vesicles cannot be explained by a reduced binding affinity of the peptide to these membranes. As shown by monitoring the leakage of a fluorescent dextran, an increase in the minimum pore size also does not explain the reduction in ANTS/DPX leakage. The data suggest that surface-associated GALA monomers or aggregates are stabilized in bilayers composed of phospholipids containing a cis unsaturation per acyl chain (DO and DPe), while transbilayer peptide insertion is reduced. GALA-induced ANTS/DPX leakage is also decreased when the vesicles contain phosphatidylethanolamine (PE). This lends further support to the suggestion that factors stabilizing the surface state of the peptide reduce its insertion and subsequent pore formation in the bilayer.     

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH₂) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.     

On the mechanism of pore formation by melittin

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Pore formation and translocation of melittin.

Melittin, a bee venom, is a basic amphiphilic peptide, which mainly acts on the lipid matrix of membranes, lysing various cells. To elucidate the molecular mechanism, we investigated its interactions with phospholipid vesicles. The peptide formed a pore with a short lifetime in the membrane, as revealed by the release of an anionic fluorescent dye, calcein, from the liposomes. Our new double-labeling method clarified that the pore size increased with the peptide-to-lipid ratio. Upon the disintegration of the pore, a fraction of the peptides translocated across the bilayer. The pore formation was coupled with the translocation, which was proved by three fluorescence experiments recently developed by our laboratory. A novel model for the melittin pore formation was discussed in comparison with other pore-forming peptides.     

Susceptibility of bacterial, eukaryotic and artificial membranes to the disruptive action of the cationic peptides Pep 5 and nisin

Abstract The cationic bactericidal peptides Pep 5 and nisin render membranes permeable to low- M _r compounds. All Gram-positive bacteria treated with these peptides showed an immediate efflux of entrapped radioactive markers. The uptake of α-[¹⁴C]methylglucoside by the phosphoenolpyruvate-dependent phosphotransferase system was stimulated by Pep 5, supporting previous results that pep 5 abolishes the membrane potential. Oxygen consumption was inhibited, presumably due to lack of ADP. Escherichia coli became sensitive to Pep 5 and nisin when the outer membrane was bypassed by osmotic shock or by formation of cytoplasmic membrane vesicles. In contrast, Mycoplasma cells and erythrocytes were unaffected by Pep 5 and nisin in concentrations up to 1 mM. Human lung fibroblasts released only small amounts of ATP when treated with Pep 5 and nisin in μM concentrations. Eukaryotic and Mycoplasma cells were disrupted more effectively by the bee venom peptide melittin, which displays overall structural similarities to Pep 5 and nisin. Various artificial membranes were not affected by Pep 5, nisin, or melittin.     

Cooperative antimicrobial action of melittin on lipid membranes: A coarse-grained molecular dynamics study

We conducted a series of coarse-grained molecular dynamics (CG-MD) simulations to investigate the complicated actions of melittin, which is an antimicrobial peptide (AMP) derived from honey bee venom, on a lipid membrane. To accurately simulate the AMP action, we developed and used a protein CG model as an extension of the pSPICA force field (FF), which was designed to reproduce several thermodynamic quantities and structural properties. At a low peptide-to-lipid (P/L) ratio (1/102), no defect was detected. At P/L = 1/51, toroidal pore formation was observed due to collective insertion of multiple melittin peptides from the N-termini. The pore formation was initiated by a local increase in membrane curvature in the vicinity of the peptide aggregate. At a higher P/L ratio (1/26), two more modes were detected, seemingly not controlled by the P/L ratio but by a local arrangement of melittin peptides: 1. Pore formation accompanied by lipid extraction by melittin peptides:a detergent-like mechanism. 2. A rapidly formed large pore in a significantly curved membrane: bursting. Thus, we observed three pore formation modes (toroidal pore formation, lipid extraction, and bursting) depending on the peptide concentration and local arrangement. These observations were consistent with experimental observations and hypothesized melittin modes. Through this study, we found that the local arrangements and population of melittin peptides and the area expansion rate by membrane deformation were key to the initiation of and competition among the multiple pore formation mechanisms.     

Effective charge of melittin upon interaction with POPC vesicles

The binding of bee venom melittin to small unilamellar vesicles and large nonsonicated multilamellar bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied by means of circular dichroism, 31P-NMR and electrophoretic mobility. The melittin binding isotherm for small unilamellar vesicles (SUV) could be described by a partition equilibrium with Kp = (6 +/- 1).10(4) M-1. Electrostatic effects were taken into account by means of the Gouy-Chapman theory. Combining the partition equilibrium with the Gouy-Chapman analysis suggested an effective charge for melittin of Zp = 1.9, which is lower than the true electric charge of 5-6. The variation of the 31P-NMR signal of SUV showed the change in potential at the phosphodiester moiety of the lipid upon addition of melittin. This potential change was lower than that for an ion with an electrical charge of 5-6 and corresponded to a charge of 1.5. Electrophoretic mobility measurements with multilamellar vesicles confirmed the charge reduction effect. These experimental results show that the use of the simple Gouy-Chapman theory requires an effective electrical charge of the melittin which is lower than the formal charge.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Kinetics of melittin induced pore formation in the membrane of lipid vesicles.

We have investigated the permeabilization of POPC unilamellar vesicle bilayers upon the addition of melittin. This process was measured in an early time range of a few minutes by means of monitoring the release of an entrapped marker, the self-quenching fluorescent dye carboxyfluorescein. Pore formation is indicated by an apparent 'all-or-none' efflux out of individual vesicles and a higher than linear dependence on melittin concentration. Applying a recently developed evaluation procedure, the data are readily converted into the gross number of pores per vesicle formed within the elapsed measuring time t. The results can be generally described in terms of a fast initial rate of pore formation that slows down to a much lower value after a period of about 1 to 2 minutes, following a single exponential time course. The three rate parameters involved are shown to be power functions of the concentration of melittin that is actually associated with the vesicle membrane. These findings are in excellent quantitative agreement with a proposed scheme of reaction steps where the formation of lipid associated peptide dimers becomes rate determining once an initial fast deposit is exhausted.     

Osmotic and pH transmembrane gradients control the lytic power of melittin.

Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes.     

Melittin-Induced Lipid Extraction Modulated by the Methylation Level of Phosphatidylcholine Headgroups

Protein- and peptide-induced lipid extraction from membranes is a critical process for many biological events, including reverse cholesterol transport and sperm capacitation. In this work, we examine whether such processes could display specificity for some lipid species. Melittin, the main component of dry bee venom, was used as a model amphipathic α-helical peptide. We specifically determined the modulation of melittin-induced lipid extraction from membranes by the change of the methylation level of phospholipid headgroups. Phosphatidylcholine (PC) bilayers were demethylated either by substitution with phosphatidylethanolamine (PE) or chemically by using mono- and dimethylated PE. It is shown that demethylation reduces the association of melittin with membranes, likely because of the resulting tighter chain packing of the phospholipids, which reduces the capacity of the membranes to accommodate inserted melittin. This reduced binding of the peptide is accompanied by an inhibition of the lipid extraction caused by melittin. We demonstrate that melittin selectively extracts PC from PC/PE membranes. This selectivity is proposed to be a consequence of a PE depletion in the surroundings of bound melittin to minimize disruption of the interphospholipid interactions. The resulting PC-enriched vicinity of melittin would be responsible for the observed formation of PC-enriched lipid/peptide particles resulting from the lipid efflux. These findings reveal that modulating the methylation level of phospholipid headgroups is a simple way to control the specificity of lipid extraction from membranes by peptides/proteins and thereby modulate the lipid composition of the membranes.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein

Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, maculatin 1.1, citropin 1.1, and aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for maculatin compared with aurein or citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, citropin and aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of maculatin, the same experimental approach was used for maculatin-Ala and maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of maculatin-Ala and maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring maculatin sequence. As observed for maculatin, the lytic action of Maculatin-Ala and maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.     

Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy

We studied the effects of melittin on various cell wall components and vesicles of various lipid compositions. To interact with the cytoplasmic membrane, melittin must traverse the cell wall, which is composed of oligosaccharides. Here, we found that melittin had a strong affinity for chitin, peptidoglycan, and lipopolysaccharide. We further examined the influence of lipid compositions on the lysis of the membranes by melittin. The result showed that melittin bound better to negatively charged than to zwitterionic lipid vesicles but was more potent at inducing leakage from zwitterionic lipid vesicles. Our studies further indicated that the oligomeric state of melittin varied between tetramers and octamers during the formation of toroidal pores. Dextran leakage experiments confirmed the formation and dimension of these toroidal pores. Finally, transmission electron microscopy revealed that melittin formed pores via peptide oligomerization by the toroidal pore-forming mechanism. The toroidal pores composed of 7-8 nm diameter rings that encircled 3.5-4.5 nm diameter cavities on zwitterionic lipid vesicles.     

Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study.

The helical order parameter of the 26-residue amphiphilic bee venom peptide melittin was measured by polarized attenuated total reflection infrared spectroscopy (ATR-IR) in dry phospholipid multibilayers (MBLs) and when bound to single supported planar bilayers (SPBs) under D2O. Melittin adopted an alpha-helical conformation in MBLs of dipalmitoyl-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), a 4:1 mixture of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and when bound to SPBs of POPC:POPG (4:1). The order parameter of the alpha-helix in the bilayers depended mainly on the type of membrane preparation, and only little on the phospholipid composition of the bilayers. On hydrated SPBs, the helical order parameter was negative, indicating that the alpha-helix long axis of melittin was preferentially oriented parallel to the plane of the supported membrane. However, in dry MBLs, the helical order parameter was positive, indicating that the alpha-helix of melittin was preferentially oriented parallel to the phospholipid fatty acyl chains. It is concluded that the orientation of melittin in membranes depends on the degree of hydration of the model membranes rather than on the technique which is used for its determination. ATR-IR spectroscopy of polypeptides in or associated with supported planar membranes in D2O may become a useful tool for the determination of their orientation in and on membranes.