Bilayer interfacial properties modulate the binding of amphipathic peptides

The free energy of transfer (DeltaG degrees ) from water to lipid bilayers was measured for two amphipathic peptides, the presequence of the mitochondrial peptide rhodanese (MPR) and melittin. Experiments were designed to determine the effects on peptide partitioning of the addition of lipids that produce structural modifications to the bilayer/water interface. In particular, the addition of cholesterol or the cholesterol analog 6-ketocholestanol increases the bilayer area compressibility modulus, indicating that these molecules modify lipid-lipid interactions in the plane of the bilayer. The addition of 6-ketocholestanol or lipids with attached polyethylene glycol chains (PEG-lipids) modify the effective thickness of the interfacial region; 6-ketocholestanol increases the width of hydrophilic headgroup region in the direction of the acyl chains whereas the protruding PEG chains of PEG-lipids increase the structural width of the headgroup region into the surrounding aqueous phase. The incorporation of PEG-lipids with PEG molecular weights of 2000 or 5000 had no appreciable effect on peptide partitioning that could not be accounted for by the presence of surface charge. However, for both MPR and melittin DeltaG degrees decreased linearly with increasing bilayer compressibility modulus, demonstrating the importance of bilayer mechanical properties in the binding of amphipathic peptides.     

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein.

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

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.     

Effect of micellar charge on the conformation and dynamics of melittin

Electrostatic interactions play a crucial role in modulating and stabilizing molecular interactions in membranes and membrane-mimetic systems such as micelles. We have monitored the change in the conformation and dynamics of the cationic hemolytic peptide melittin bound to micelles of various charge types, utilizing fluorescence and circular dichroism (CD) spectroscopy. The sole tryptophan of melittin displays a red-edge excitation shift (REES) of 3-6 nm when bound to anionic, nonionic, and zwitterionic micelles. This suggests that melittin is localized in a restricted environment, probably in the interfacial region of the micelles, and this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state tryptophan in melittin. Further, the rotational mobility of melittin is considerably reduced in these micelles and is found to be dependent on the surface charge of micelles. Interestingly, our results show that melittin does not partition into cetyltrimethylammonium bromide (CTAB) micelles owing to electrostatic repulsion between melittin and CTAB micelles, both of which carry a positive charge. In addition, the fluorescence lifetime of melittin is modulated in micelles of different charge types. The lowest mean fluorescence lifetime is observed in the case of melittin bound to anionic sodium dodecyl sulfate (SDS) micelles. CD spectroscopy shows that micelles induce significant helicity to melittin, with maximum helicity being induced in the case of melittin bound to SDS micelles. Fluorescence quenching measurements using the neutral aqueous quencher acrylamide show differential accessibility of melittin in various types of micelles. Taken together, our results show that micellar surface charge can modulate the conformation and dynamics of melittin. These results could be relevant to understanding the role of the surface charge of membranes in the interaction of membrane-active, amphiphilic peptides with membranes.     

Melittin: structure, properties, interaction with a membrane

The paper is concerned with the analysis of the present data as to the structure and properties of melittin, a biologically active peptide of bee venom, able to be inserted spontaneously into the natural and synthetic membranes and to destruct cells in micromolar concentrations. This property is a result of the peptide structure peculiarities. Attention is focused on the mechanism of melittin insertion into the phospholipid bilayer as well as on the possibility of its use to study the nature of protein-lipid interactions and formation of ionic channels.     

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Membrane fusion activity of succinylated melittin is triggered by protonation of its carboxyl groups

The membrane fusion activity of melittin and its succinylated derivative was studied as a function of pH by the transfer of spin-labeled phosphatidylcholine as well as by internal content mixing and electron microscopy. The protonation process of the carboxyl groups introduced into melittin was studied by 13C NMR spectroscopy using derivative prepared with [1,4(-13)C]succinic anhydride. Melittin causes fusion of sonicated phosphatidylcholine vesicles in a wide range of pH. In marked contrast, melittin with all four amino groups succinylated induces fusion only at acidic pH lower than 5.2, with the maximum at pH 5.1. The fusion reactions are very rapid, reaching a saturation level within 1 min. The fusion efficiency depends on the peptide-to-phospholipid ratio in the reaction mixture. Trypsinized succinylated melittin, which has lost the four positively charged C-terminal residues, causes aggregation of vesicles at acidic pH but cannot induce fusion. The 13C NMR peaks for the carboxyl and carbonyl groups of succinylated melittin shifted to higher field as the pH was lowered. The pKa value of the four carboxyl groups was obtained as 5.19 and 4.83 in the presence and absence of vesicles, respectively. The pKa value in the presence of vesicles agrees quite well with the half-maximal pH for fusion of 5.15, indicating that the fusion activity is triggered by protonation of the carboxyl groups in the hydrophobic segment of the peptide. The higher shift of pKa value in the presence of vesicles can be due to stabilization of the protonated form by entrance into lipid bilayer hydrocarbon layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic selectivity of melittin-modified flat lipid bilayers]

It has been established in experiments with the bilayer lipid membranes (BLM) that at pH greater than 6.6 the melittin pores are cation-selective and at lower pH they are more selective by anions. The property of melittin pores is shown to be provided by the amino group of the N-terminal glycine residue. The selectivity of melittin-containing membranes may be controlled by the transmembrane potential, the cross-section of water pores being changed. The data obtained are explained within the alimethicine-like model.     

A combined study of aggregation, membrane affinity and pore activity of natural and modified melittin

The pore activity of melittin and several chemically modified derivatives has been investigated using conductance measurements on planar lipid bilayers and marker release from small unilamellar vesicles. The modifications included N-terminal formylation, acetylation, succinylation and modification of the tryptophan residue. All of the compounds showed bilayer permeabilizing properties, though quantitative differences were evident. These comprised changes in the voltage dependence of the conductance, in the single-pore kinetics, in the concentration of aqueous peptide required to induce a given pore activity and in the apparent 'molecularity' reflected by the power law of its concentration dependence. A strong tendency for disrupting bilayers was not always correlated with strong pore activity. For a better understanding of these results, measurements of pore activity were complemented by studying the aggregation behavior in solution and the water-membrane partition equilibrium. Modifications of charged residues gave rise to significant changes in the aggregation properties, had virtually no influence on the partition coefficient. The latter decreased strongly, however, as a result of tryptophan modification. Analysis of the isotherms was consistent with the assumption that the arginine residues in melittin do not contribute very much to charge accumulation at the immediate membrane/water interface.     

Kinetics and mechanism of hemolysis induced by melittin and by a synthetic melittin analogue.

The cytotoxic peptide from honeybee venom, melittin, and a synthetic peptide analogue of it lyse human erythrocytes in a biphasic process. The kinetics of the lysis in 0.30 M sucrose, 0.01 M sodium phosphate, pH 7.30 at 4 degrees C were investigated. Our results show that melittin rapidly binds to the outer surface of the erythrocyte membrane, and the surface-bound monomers produce transient openings through which approximately 40 hemoglobin molecules can escape. Concomitantly, the melittin loses its ability to effect the process, presumably by translocation through the bilayer. The half-life for this process is 1.2 min. In a much slower process, dimers of this internalized melittin again produce transient membrane openings in a steady state. On a molar basis, the synthetic peptide analogue produces a fast process comparable to that caused by melittin, but is more efficient in the slow phase. Escape of hemoglobin and of carbonic anhydrase through the openings is diffusion controlled. These results suggest that the functional units necessary for the activity of melittin-like cytotoxic peptides are a 20 amino acid amphiphilic alpha-helix with a hydrophobic:hydrophilic ratio greater than 1 and a short segment with a high concentration of positive charges.     

Modulation of melittin-induced lysis by surface charge density of membranes.

Phosphorus NMR spectroscopy was used to characterize the importance of electrostatic interactions in the lytic activity of melittin, a cationic peptide. The micellization induced by melittin has been characterized for several lipid mixtures composed of saturated phosphatidylcholine (PC) and a limited amount of charged lipid. For these systems, the thermal polymorphism is similar to the one observed for pure PC: small comicelles are stable in the gel phase and extended bilayers are formed in the liquid crystalline phase. Vesicle surface charge density influences strongly the micellization. Our results show that the presence of negatively charged lipids (phospholipid or unprotonated fatty acid) reduces the proportion of lysed vesicles. Conversely, the presence of positively charged lipids leads to a promotion of the lytic activity of the peptide. The modulation of the lytic effect is proposed to originate from the electrostatic interactions between the peptide and the bilayer surface. Attractive interactions anchor the peptide at the surface and, as a consequence, inhibit its lytic activity. Conversely, repulsive interactions favor the redistribution of melittin into the bilayer, causing enhanced lysis. A quantitative analysis of the interaction between melittin and negatively charged bilayers suggests that electroneutrality is reached at the surface, before micellization. The surface charge density of the lipid layer appears to be a determining factor for the lipid/peptide stoichiometry of the comicelles; a decrease in the lipid/peptide stoichiometry in the presence of negatively charged lipids appears to be a general consequence of the higher affinity of melittin for these membranes.     

Interaction of melittin with phosphatidylcholine membranes. Binding isotherm and lipid head-group conformation

The binding of melittin to nonsonicated bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with an ultracentrifugation assay and with 2H and 31P nuclear magnetic resonance. Melittin binding could best be described by a partition equilibrium with Kp = (2.1 +/- 0.2) X 10(3) M-1, measuring the binding isotherm in the concentration range of 0-100 microM melittin and taking into account electrostatic effects by means of the Gouy-Chapman theory. This partition coefficient is smaller than that deduced for small sonicated vesicles and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium magnetic resonance revealed a conformational change of the phosphocholine head group upon melittin binding. The quadrupole splittings of the alpha and beta segments of the choline head group varied linearly with the amount of bound melittin but in opposite directions; i.e., the alpha splitting decreased, and the beta splitting increased. This conformational change is not specific to melittin but is a response of the phosphocholine head group to positive membrane surface charges in general. Quantitatively, melittin is one of the most efficient head-group modulators, the efficiency per unit charge comparable to that of charged local anesthetics or hydrophobic ions.     

Protein-induced membrane disorder: a molecular dynamics study of melittin in a dipalmitoylphosphatidylcholine bilayer

A molecular dynamics simulation of melittin in a hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer was performed. The 19, 000-atom system included a 72-DPPC phospholipid bilayer, a 26-amino acid peptide, and more than 3000 water molecules. The N-terminus of the peptide was protonated and embedded in the membrane in a transbilayer orientation perpendicular to the surface. The simulation results show that the peptide affects the lower (intracellular) layer of the bilayer more strongly than the upper (extracellular) layer. The simulation results can be interpreted as indicating an increased level of disorder and structural deformation for lower-layer phospholipids in the immediate vicinity of the peptide. This conclusion is supported by the calculated deuterium order parameters, the observed deformation at the intracellular interface, and an increase in fractional free volume. The upper layer was less affected by the embedded peptide, except for an acquired tilt relative to the bilayer normal. The effect of melittin on the surrounding membrane is localized to its immediate vicinity, and its asymmetry with respect to the two layers may result from the fact that it is not fully transmembranal. Melittin's hydrophilic C-terminus anchors it at the extracellular interface, leaving the N-terminus "loose" in the lower layer of the membrane. In general, the simulation supports a role for local deformation and water penetration in melittin-induced lysis. As for the peptide, like other membrane-embedded polypeptides, melittin adopts a significant 25 degree tilt relative to the membrane normal. This tilt is correlated with a comparable tilt of the lipids in the upper membrane layer. The peptide itself retains an overall helical structure throughout the simulation (with the exception of the three N-terminal residues), adopting a 30 degree intrahelical bend angle.     

Incorporation of highly purified melittin into phosphatidylcholine bilayer vesicles

Melittin free of phospholipase A2 was prepared. In the absence of salt this highly pure protein starts to aggregate in solution at a protein concentration of Cp greater than 10(-3) M. In high salt solution (2 M) aggregation starts at Cp greater than 10(-6) M. This was determined from the blue shift of the intrinsic fluorescence of the protein. Reinvestigation of the quenching behaviour clearly shows that self-aggregation cannot be deduced from quenching experiments using nitrate or 2,2,6,6-tetramethylpiperidine-1-oxyl as quencher. The incorporation of melittin into phosphatidylcholine bilayer vesicles was studied by fluorescence quenching and by energy-transfer experiments using 2- and 6-anthroyloxypalmitic acid as acceptor and peptide tryptophan as donor. Incorporation of melittin into small unilamellar vesicles was found to be reduced below the lipid phase transition temperature, Tt, whereas it incorporates and distributes more randomly above Tt. Cooling the temperature below Tt after incubation at T greater than Tt leads to a deeper incorporation of the peptide into the lipid bilayer due to electrostatic interaction between the lipid phosphate groups and the positively charged amino acids. This stabilizing effect is lost above Tt and melittin is extruded to the polar phase. Quenching experiments support this finding. EPR measurements clearly demonstrate that even in the presence of high amounts of melittin up to 10 mol% with respect to the lipid broadening of the phase transition curves was only observed with fatty acid spin labels, where the doxyl group is localized near the bilayer surface. The order degree of the inner part of the bilayer remains almost unchanged even in the presence of high melittin content.     

Influence of lipid chain unsaturation on melittin-induced micellization.

It is well known that melittin, an amphipathic helical peptide, causes the micellization of phosphatidylcholine vesicles. In the present work, we conclude that the extent of micellization is dependent on the level of unsaturation of the lipid acyl chains. We report the results obtained on two systems: dipalmitoylphosphatidylcholine (DPPC), containing 10(mol)% saturated or unsaturated fatty acid (palmitic, oleic, or linoleic), and DPPC, containing 10(mol)% positively charged diacyloxy-3-(trimethylammonio)propane bearing palmitic or oleic acyl chains. For both systems, the presence of unsaturation in the lipid acyl chains inhibits melittin-induced micellization. Conversely, the addition of saturated palmitic acid to the DPPC matrix enhances the micellization. This modulation is proposed to be associated with the cohesion of the hydrophobic core. When the lipid chain packing of the gel-phase bilayer is already perturbed by the presence of unsaturation, it seems easier for the membrane to accommodate melittin at the interface, and the distribution of the peptide in the bilayer could be the origin of the inhibition of the micellization. The cohesion of the apolar core is shown to play an unquestionable role in melittin-induced micellization; however, this contribution does not appear to be as important as the electrostatic interactions between melittin and positively or negatively charged lipids.     

The presence of PEG-lipids in liposomes does not reduce melittin binding but decreases melittin-induced leakage.

Poly(ethyleneglycol) (PEG), anchored at the surface of liposomes via the conjugation to a lipid, is commonly used for increasing the liposome stability in the blood stream. In order to gain a better understanding of the protective properties of interfacial polymers, we have studied the binding of melittin to PEG-lipid-containing membranes as well as the melittin-induced efflux of a fluorescent marker from liposomes containing PEG-lipids. We examined the effect of the polymer size by using PEG with molecular weights of 2000 and 5000. In addition, we studied the role of the anchoring lipid by comparing PEG conjugated to phosphatidylethanolamine (PE) which results in a negatively charged PEG-PE, with PEG conjugated to ceramide (Cer) which provides the neutral PEG-Cer. Our results show that interfacial PEG does not prevent melittin adsorption onto the interface. In fact, PEG-PE promotes melittin binding, most likely because of attractive electrostatic interactions with the negative interfacial charge density of the PEG-PE-containing liposomes. However, PEG-lipids limit the lytic potential of melittin. The phenomenon is proposed to be associated with the change in the polymorphic tendencies of the liposome bilayers. The present findings reveal that the protective effect associated with interfacial hydrophilic polymers is not universal. Molecules like melittin can sense surface charges borne by PEG-lipids, and the influence of PEG-lipids on liposomal properties such as the polymorphic propensities may be involved in the so-called protective effect.     

The presence of PEG-lipids in liposomes does not reduce melittin binding but decreases melittin-induced leakage

Poly(ethyleneglycol) (PEG), anchored at the surface of liposomes via the conjugation to a lipid, is commonly used for increasing the liposome stability in the blood stream. In order to gain a better understanding of the protective properties of interfacial polymers, we have studied the binding of melittin to PEG-lipid-containing membranes as well as the melittin-induced efflux of a fluorescent marker from liposomes containing PEG-lipids. We examined the effect of the polymer size by using PEG with molecular weights of 2000 and 5000. In addition, we studied the role of the anchoring lipid by comparing PEG conjugated to phosphatidylethanolamine (PE) which results in a negatively charged PEG-PE, with PEG conjugated to ceramide (Cer) which provides the neutral PEG-Cer. Our results show that interfacial PEG does not prevent melittin adsorption onto the interface. In fact, PEG-PE promotes melittin binding, most likely because of attractive electrostatic interactions with the negative interfacial charge density of the PEG-PE-containing liposomes. However, PEG-lipids limit the lytic potential of melittin. The phenomenon is proposed to be associated with the change in the polymorphic tendencies of the liposome bilayers. The present findings reveal that the protective effect associated with interfacial hydrophilic polymers is not universal. Molecules like melittin can sense surface charges borne by PEG-lipids, and the influence of PEG-lipids on liposomal properties such as the polymorphic propensities may be involved in the so-called protective effect.     

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.