Different states of self-association of melittin in phospholipid bilayers

Melittin is known to self-associate as tetramers in solutions of high ionic strength. Here, an N-bromosuccinimide oxidized-Trp₁₉ melittin is prepared. This derivative can act as an acceptor of the fluorescence of native melittin and is used in order to observe a possible self-association of melittin in phospholipid bilayers.Resonance energy transfer was shown to occur in solutions of high ionic strength, showing that oxidized melittin can associate with native melittin.In phospholipid bilayers, no association is detected in the absence of NaCl. In its presence, an equilibrium between monomeric melittin and oligomeric species is observed. These species are not dimers, but any other degree of association may account for our experimental results. Significant differences in characteristic transfer efficiency reveal differences in the structure of these oligomers according to the length or state of phospholipids (fluid or at the transition temperature). These bound complexes are also different from the soluble hetero-oligomer.Some models of bound complexes are proposed which may explain the leakage and the further disruption of vesicles or cells induced by melittin.Abbreviations NBS N-bromosuccinimide - NATA N-acetyl tryptophanamide - DMPC dimyristoyl phosphatidylcholine - DPPC dipalmitoyl phosphatidylcholine - PG phosphatidylglycerol - EPC egg phosphatidylcholine - O-melittin oxindole-melittin - RET resonance energy transfer - EDTA ethylene diamine tetracetic acid - Mel melittin     

Real-time structural investigation of a lipid bilayer during its interaction with melittin using sum frequency generation vibrational spectroscopy

Interactions between membrane bilayers and peptides/proteins are ubiquitous throughout a cell. To determine the structure of membrane bilayers and the associated peptides/proteins, model systems such as supported lipid bilayers are often used. It has been difficult to directly investigate the interactions between a single membrane bilayer and peptides/proteins without exogenous labeling. In this work we demonstrate that sum frequency generation vibrational spectroscopy can be employed to study the interactions between peptides/proteins and a single lipid bilayer in real time, in situ, and without exogenous labeling. Using melittin and a dipalmitoyl phosphatidylglycerol bilayer as a model system, we monitored the C-H and C-D stretching signals from isotopically symmetric or asymmetric dipalmitoyl phosphatidylglycerol bilayers during their interaction with melittin. It has been found that the extent and kinetics of bilayer perturbation induced by melittin are very sensitive to melittin concentration. Such concentration dependence is correlated to melittin's mode of action. Melittin is found to function via the early and late stage of the carpet model at low and high concentrations, respectively, whereas the toroidal model is probable at intermediate concentrations. This research illustrates the potential of sum frequency generation as a biophysical technique to monitor individual leaflet structure of lipid bilayers in real time during their interactions with biomolecules.     

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.     

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.     

Acyl Transfer from Membrane Lipids to Peptides Is a Generic Process

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography–mass spectrometry analysis of peptide–lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH 7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3 h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.     

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.     

Measurement of the affinity of melittin for zwitterionic and anionic membranes using immobilized lipid biosensors.

The binding of melittin to zwitterionic dimyristyphosphatidylcholine (DMPC) and anionic dimyristylphosphatidylglycerol (DMPG) was analysed using two different immobilized model membrane systems. The first system used surface plasmon resonance (SPR), which monitors the real-time binding of peptides to an immobilized hybrid bilayer. SPR experiments reflected a stronger binding of melittin for DMPG than for DMPC, while kinetic analysis suggested the existence of at least two distinct binding steps. The second lipid biosensor system involved an immobilized phospholipid monolayer covalently attached to a microporous silica surface. The binding of melittin to the immobilized monolayer was then monitored using dynamic elution chromatography with varied methanol concentrations to analyse the binding of melittin to DMPC and DMPG. The nonlinear binding behaviour observed for melittin with the phosphatidylcholine (PC) and phosphatidylglycerol (PG) monolayers compared with the linear retention plots and Gaussian peak shapes observed for the control molecule demonstrated that melittin undergoes significant conformational and orientational changes upon binding to the immobilized PC and PG ligands. The dependence of log k' on per cent methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while other forces, presumably electrostatic in nature, also made a contribution to the affinity of the peptides for the lipid monolayer, particularly at lower temperatures. The complementary use of these two lipid biosensors thus allows the role of hydrophobic and electrostatic forces in peptide-membrane interactions to be studied.     

Quinidine and melittin both decrease the fluidity of liver plasma membranes and both inhibit hormone-stimulated adenylate cyclase activity

Increasing concentrations of either quinidine or melittin gave a dose-dependent inhibition of both the glucagon- and fluoride-stimulated activities of adenylate cyclase in the liver plasma membranes. At similar concentrations these agents increased the order of liver plasma membranes as detected by a fatty acid ESR probe, doxyl stearic acid. This increase in bilayer order (decrease in 'fluidity') is suggested to explain the inhibitory action of quinidine on adenylate cyclase activity but only in part contributes to the inhibitory action of melittin on adenylate cyclase. Arrhenius plots of fluoride-stimulated activity became non-linear in the presence of either quinidine or melittin, with a single well-defined break occurring at around 12 degrees C in each instance. Arrhenius plots of the glucagon-stimulated activity also exhibited such a novel break at around 12 degrees C when either quinidine or melittin were present as well as exhibiting a break at around 28 degrees C, as was seen in the absence of these ligands. The fatty acid spin probe inserted into liver plasma membranes detected a novel lipid phase separation occurring at around 12 degrees C when either quinidine or melittin was present and showed that the lipid phase separation occurring at around 28 degrees C in native membranes was apparently unaffected by these ligands.     

Phospholipase A2 activation by melittin causes amylase release from exocrine pancreas

Phospholipase A2-induced deacylation of membrane phospholipids is associated with changes in membrane fluidity. The importance of this reaction in the pancreatic amylase secretory process was tested using melittin, a phospholipase A2 stimulating peptide. Phospholipase A2 activity (using [3H]arachidonic acid release as an index) and amylase secretion were both increased in a time- and concentration-dependent manner by melittin. Phospholipids prelabelled with [3H]oleic acid or [14C]linoleic acid also released radioactive free fatty acids in response to melittin. Prostaglandin synthesis was not involved in the melittin response, since inhibitors of arachidonic acid oxidation (indomethacin, 5,8,11,14-eicosatetraynoic acid) did not alter the ability of melittin to release [3H]arachidonic acid or amylase. When melittin was co-applied with carbachol, cholecystokinin octapeptide, or vasoactive intestinal peptide, amylase secretion was additive. The effect of melittin on both fatty acid and amylase release was dependent on extracellular calcium, though melittin's effects were not dependent on the intracellular accumulation of second messengers such as calcium or cAMP. The data suggest that activation of phospholipase A2 by melittin results in the triggering of the secretory process in exocrine pancreas by a different mechanism than that for other pancreatic secretagogues.     

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.     

Hemolytic and antimicrobial activities of the twenty-four individual omission analogues of melittin

Although melittin's hemolytic activity has been extensively studied, the orientation of membrane-bound melittin remains uncertain. We have investigated the effect of individually omitted amino acid residues on melittin's activity and related these results to the existing models of melittin-membrane interaction. The extent of hemolysis of the omission analogues closely followed the four known conformational regions of melittin: omission of any of the residues making up the two alpha-helical regions decreased the hemolytic activity relative to melittin, while omission of any of the residues making up the "hinge" or the C-terminal regions had little or no effect. Our results correlate best with a proposed model in which melittin initially forms "holes" in the membrane, resulting in an initial rapid loss of hemoglobin; the membrane-bound melittin is then internalized into the membrane, resulting in a later slow phase of hemoglobin loss. It was also found that induced structural effects caused by peptide-lipid interactions could be studied by using RP-HPLC, with an excellent correlation found between the retention times of the individual omission analogues and their hemolytic activities.     

The influence of cytotoxins from Central Asian cobra venom and melittin from bee venom on the thermodynamic properties of phospholipid bilayer

The interaction of cytotoxin Vc1 and Vc5 from Central Asian cobra and melittin from the bee venom with multilayer liposomes prepared from dimiristoylphosphatidylcholine with an addition of phosphatidic acid have been studied by the method of differential scanning calorimetry. Incorporation of Vc1, Vc5 and melittin into the lipid resulted in pronounced changes in the thermodynamic properties of the lipid. Polypeptides studied induced lateral phase separation in the lipid. Interaction between molecules of the toxins and the lipid resulted in the formation of a new lipid phase characterized by a higher melting temperature and lower phase transition enthalpy.     

The structure of melittin in the form I crystals and its implication for melittin's lytic and surface activities.

Melittin from bee venom is water-soluble, yet integrates into membranes and lyses cells. Each melittin chain consists of 26 amino acid residues and in aqueous salt solutions it exists as a tetramer. We have determined the molecular structure of the tetramer in two crystal forms grown from concentrated salt solutions. In both crystal forms the melittin polypeptide is a bent alpha-helical rod, with the "inner" surface largely consisting of hydrophobic sidechains and the "outer" surface consisting of hydrophilic side chains. Thus, the helix is strongly amphiphilic. In the tetramer, four such helices contribute their hydrophobic side chains to the center of the molecule. The packing of melittin tetramers is also very similar in the two crystal forms: they are packed in planar layers with the outsides forming hydrophilic surfaces and the insides (the centers of melittin tetramers) forming a hydrophobic surface. We suggest that the surface activity of melittin can be rationalized in terms of these surfaces. The lytic activity of melittin can also be interpreted in terms of the molecular structure observed in the crystals: the hydrophobic inner surface of a melittin helix may integrate into the apolar region of a bilayer with the helix axis approximately parallel to the plane of the bilayer, and with the hydrophilic surface exposed to the aqueous phase. This integration would be expected to disrupt the bilayer because of melittin helix would penetrate only a short distance into it. Additionally, the integration of melittin from one side of a bilayer would produce a surface area difference across the bilayer, perhaps leading to lysis. In this view, melittin is distinct from membrane proteins that penetrate evenly into both leaflets of a bilayer or exactly halfway through a bilayer, and hence we refer to melittin as a surface-active protein.     

The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus

Pulse-chase experiments with [2-3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol leads to (glycerophospho)25-diglucosyldiacylglycerol + 25 diacylglycerol. Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor. Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative. In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.     

Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells

In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp. strain A007. When cells of B. subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol. Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity. The [32P]phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B. subtilis W23. A unique metabolism of phosphatidylglycerol was found in Bacillus sp. strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B. subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate. These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover.     

How lipids influence the mode of action of membrane-active peptides

The human, multifunctional peptide LL-37 causes membrane disruption by distinctly different mechanisms strongly dependent on the nature of the membrane lipid composition, varying not only with lipid headgroup charge but also with hydrocarbon chain length. Specifically, LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged phosphatidylglycerol (PG) model membranes, where the hydrocarbon chains are shielded from water by the peptide. In turn, LL-37 leads to a disintegration of the lamellar organization of zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) into disk-like micelles. Interestingly, interdigitation was also observed for the longer-chain C18 and C20 PCs. This dual behavior of LL-37 can be attributed to a balance between electrostatic interactions reflected in different penetration depths of the peptide and hydrocarbon chain length. Thus, our observations indicate that there is a tight coupling between the peptide properties and those of the lipid bilayer, which needs to be considered in studies of lipid/peptide interaction. Very similar effects were also observed for melittin and the frog skin peptide PGLa. Therefore, we propose a phase diagram showing different lipid/peptide arrangements as a function of hydrocarbon chain length and LL-37 concentration and suggest that this phase diagram is generally applicable to membrane-active peptides localized parallel to the membrane surface.     

How lipids influence the mode of action of membrane-active peptides

The human, multifunctional peptide LL-37 causes membrane disruption by distinctly different mechanisms strongly dependent on the nature of the membrane lipid composition, varying not only with lipid headgroup charge but also with hydrocarbon chain length. Specifically, LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged phosphatidylglycerol (PG) model membranes, where the hydrocarbon chains are shielded from water by the peptide. In turn, LL-37 leads to a disintegration of the lamellar organization of zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) into disk-like micelles. Interestingly, interdigitation was also observed for the longer-chain C18 and C20 PCs. This dual behavior of LL-37 can be attributed to a balance between electrostatic interactions reflected in different penetration depths of the peptide and hydrocarbon chain length. Thus, our observations indicate that there is a tight coupling between the peptide properties and those of the lipid bilayer, which needs to be considered in studies of lipid/peptide interaction. Very similar effects were also observed for melittin and the frog skin peptide PGLa. Therefore, we propose a phase diagram showing different lipid/peptide arrangements as a function of hydrocarbon chain length and LL-37 concentration and suggest that this phase diagram is generally applicable to membrane-active peptides localized parallel to the membrane surface.     

The aggregation state of mellitin in lipid bilayers. An energy transfer study

We used fluorescence non-radiative energy transfer to measure the self-association of melittin in solution and when bound to lipid bilayers. Energy transfer occurred from the tryptophan residue of unlabeled melittin to an N-methyl anthraniloyl residue covalently bound to a basic lysine residue on melittin. The extent of energy transfer from tryptophan to the label was found to increase severalfold upon the salt-induced tetramerization of melittin. When bound to vesicles of dimyristoyl-L-alpha-phosphatidylcholine, the extent of energy transfer was found to be equivalent to that of monomeric melittin, irrespective of the presence of monomeric or tetrameric melittin in the aqueous phase. We conclude that membrane-bound melittin is monomeric.     

Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin

Affinity chromatography, fluorescence and circular dichroism spectroscopy methods have been used to study the interaction of melittin, a 26-residue peptide from bee venom, with Ca2(+)-binding alpha-lactalbumin from human milk. It has been revealed that melittin binds to the apo- and acidic states of alpha-lactalbumin while the presence of Ca2+ makes the interaction essentially weaker. The association constant for the complex of melittin with apo-alpha-lactalbumin determined from spectropolarimetric melittin-titration data is 2 X 10(7) M-1. The complexation of alpha-lactalbumin with melittin decreases its affinity to Ca2+ by three orders of magnitude. The interaction of apo-alpha-lactalbumin with melittin causes some changes in the environment of its aromatic amino acid residues and drastically alters the conformation of melittin, increasing its alpha-helical content but leaving its single tryptophan residue accessible to water. In the case of the acidic state of alpha-lactalbumin the interaction does not induce an increase in alpha-helical content of melittin.     

On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii.

About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A₂ from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglycerol pool. However, modification of the membrane proteins with 2,4,6-trinitrobenzenesulfonic acid or glutaraldehyde did make an additional 70% of this protected pool of phosphatidylglycerol accessible to phospholipase A₂. Complete hydrolysis of phosphatidylglycerol at low incubation temperatures was achieved only after heat treatment of the membranes which resulted in an extensive aggregation of intrinsic membrane proteins as visualized by freeze-etch electron microscopy. Phospholipase A₂ from bee venom was more effective in hydrolyzing phosphatidylglycerol at low temperature than the pancreatic enzyme. These results show that the inaccessibility of phosphatidylglycerol is not due to resealing of isolated membranes, the presence of a crystalline phase in the membrane lipids, or a shielding effect of surface proteins. The protection against hydrolysis may be due to an interaction of phosphatidylglycerol with intrinsic membrane proteins which is stabilized at low temperatures. Increasing the temperature favors the exchange of protein-bound phosphatidylglycerol with other membrane lipids resulting in complete hydrolysis.