melitten and 1-2-oleoylphosphatidylcholine

Many fundamental biological processes occur on cell membranes, and a typical example is the membrane permeabilization by peptides for an antimicrobial purpose. Previous studies of the underlying mechanism mostly focus on structural changes of membranes and peptides during their interactions. Herein, from a new perspective of single-molecule dynamics, the real-time three-dimensional motions of individual phospholipid and peptide molecules were monitored, and specifically, their correlation with the membrane poration function of melittin, a most representative natural antimicrobial peptide, was studied. We found that the adsorption and accumulation of melittin on the membrane surface significantly sped up the lateral diffusion of lipids surrounding the peptides, which in turn facilitated the peptide insertion at such heterogeneous regions. A unique "U"-bending pathway of melittin during membrane insertion and the ultimate formation of toroidal pores with dynamical translocations of peptides and lipids with several metastable states between the two leaflets of bilayer were observed.

Topics: Adsorption; Diffusion; Lipid Bilayers; Melitten; Molecular Dynamics Simulation; Phosphatidylcholines; Single Molecule Imaging; Unilamellar Liposomes

This article presents coarse-grained molecular dynamics simulations of pore-forming antimicrobial peptide melittin and its interactions with vesicles composed of a mixture of zwitterionic and anionic phospholipids. Besides creating holes in the membrane, the adsorption of melittin also induces vesicle budding, which can develop into vesiculation at high peptide concentrations, as well as vesicle invagination, which can eventually result in a corrugated membrane surface. These rich morphology changes are mediated by the curvature of the vesicles and the peptide concentration. Highly curved vesicles favor the recruitment of melittins with a higher density of binding sites. The peptides mainly penetrate into the membrane surface in monomers via hydrophobic interaction. Lowly curved vesicles recruit melittins with a low density of binding sites. Surplus peptides are prone to form oligomers and shallowly adsorb on the surface of membrane via electrostatic interaction. The penetration of monomers induces membrane pore formation and positive membrane curvature, which promote vesicle budding. The adsorption of oligomers induces negative membrane curvature, which promotes vesicle invagination. This work demonstrates that antimicrobial peptides adopt multiple actions to destroy bacterial membranes.

Topics: Anti-Bacterial Agents; Binding Sites; Cell Membrane; Melitten; Molecular Dynamics Simulation; Peptide Fragments; Phosphatidylcholines; Phosphatidylglycerols; Protein Conformation; Protein Interaction Domains and Motifs; Unilamellar Liposomes

Quantitative characterization of membrane defects (pores) is important for elucidating the molecular basis of many membrane-active peptides. We study kinetic defects induced by melittin in vesicular and planar lipid bilayers. Fluorescence spectroscopy measurements indicate that melittin induces time-dependent calcein leakage. Solution atomic force microscopy (AFM) is used to visualize melittin-induced membrane defects. After initial equilibration, the most probable defect radius is ∼3.8 nm in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) bilayers. Unexpectedly, defects become larger with longer incubation, accompanied by substantial shape transformation. The initial defect radius is ∼4.7 nm in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. Addition of 30 mol % cholesterol to DOPC bilayers suppresses defect kinetics, although the inhibitory impact is negated by longer incubation. Overall, the kinetic rate of defect development follows DLPC > DOPC > DOPC/cholesterol. Kinetic defects are also observed when anionic lipids are present. Based on the observation that defects can occupy as large as 40% of the bilayer surface, we propose a kinetic defect growth model. We also study the effect of melittin on the phase behavior of DOPC/egg-sphingomyelin/cholesterol bilayers. We find that melittin initially suppresses or eliminates liquid-ordered (Lo) domains; Lo domains gradually emerge and become the dominant species with longer incubation; and defects in phase-coexisting bilayers have a most probable radius of ∼5 nm and are exclusively localized in the liquid-disordered (Ld) phase. Our experimental data highlight that melittin-induced membrane defects are not static; conversely, spontaneous defect growth is intrinsically associated with membrane permeabilization exerted by melittin.

Topics: Cholesterol; Kinetics; Lipid Bilayers; Melitten; Microscopy, Atomic Force; Phosphatidylcholines; Spectrometry, Fluorescence

The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated.

Topics: Aluminum Oxide; Cell Membrane; Lipid Bilayers; Melitten; Microscopy, Fluorescence; Phosphatidylcholines; Protein Transport; Spectrum Analysis; Unilamellar Liposomes

We performed a series of coarse-grained computer simulations in order to study how the placement of melittin and magainin-h2 antimicrobial peptides on the surface of the bilayer changes the local pressure profiles in the bilayer. The simulations were done using the NPT ensemble when the total stress on the bilayer was zero and also using the NP(z)AT ensemble, with a nonzero total stress. In the NPT ensemble, although the total stress was zero, each leaflet of the bilayer experienced a nonzero stress, and the stresses are equal by magnitude, but opposite in their direction. The observed stresses acting on the monolayers may cause the rupture of the monolayers to release the stress. Our simulations were done at different peptide to lipid ratio (P/L). When the P/L ratio was 1/50 there was no large difference in the local pressure profile for bilayers with melittin versus bilayers with magainin-h2. When simulations were performed in the NP(z)AT ensemble at P/L = 3/100 we observed a large difference in the pressure profiles in the bilayers with melittin peptides compared to the bilayer with magainin-h2. The observed in this case difference in stress may explain the difference in actions of melittin and magainin at high P/L.

Topics: Adsorption; Animals; Antimicrobial Cationic Peptides; Bees; Kinetics; Lipid Bilayers; Magainins; Melitten; Molecular Dynamics Simulation; Phosphatidylcholines; Pressure; Thermodynamics; Xenopus laevis; Xenopus Proteins

The distribution of peptide conformations in the membrane interface is central to partitioning energetics. Molecular-dynamics simulations enable characterization of in-membrane structural dynamics. Here, we describe melittin partitioning into dioleoylphosphatidylcholine lipids using CHARMM and OPLS force fields. Although the OPLS simulation failed to reproduce experimental results, the CHARMM simulation reported was consistent with experiments. The CHARMM simulation showed melittin to be represented by a narrow distribution of folding states in the membrane interface.

Topics: Lipid Bilayers; Melitten; Molecular Dynamics Simulation; Phosphatidylcholines; Protein Conformation

Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the "increasing membrane area", "phased shrinkage", or "solubilization" of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, β-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions.

Topics: Animals; Chemical Phenomena; Circular Dichroism; Hydrophobic and Hydrophilic Interactions; Insect Proteins; Kinetics; Lipid Bilayers; Liposomes; Melitten; Membrane Proteins; Membranes; Microscopy, Electron, Transmission; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids; Protein Structure, Secondary; Solubility; Surface Properties; Tryptophan

The innate reactivity of the peptide melittin (H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH(2)) towards membrane lipids has been explored using LC-MS methods. The high sensitivity afforded by LC-MS analysis enabled acyl transfer to the peptide to be detected, within 4 h, from membranes composed of phosphocholines (PCs). Acyl transfer from PCs was also observed from mixtures of PC with phosphoserine (PS) or phosphoglycerol (PG). In the latter case, transfer from PG was also detected. The half-lives for melittin conversion varied between 24 h and 75 h, being fastest for POPC and slowest for DOPC/DMPG mixtures. The order of reactivity for amino groups on the peptide was N-terminus > K23 ≫ K21 > K7. Products arising from double-acylation of melittin were detected as minor components, together with a putative component derived from transesterification involving S18 of the peptide.

Topics: Amino Acid Sequence; Chromatography, Liquid; Mass Spectrometry; Melitten; Membrane Lipids; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Phospholipids

Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of engineered nanomaterials (ENMs) with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assess the effect of varying the surface functionality and concentration of 10-nm-diameter gold (Au) and titanium dioxide (TiO(2)) ENMs on the disruption of negatively charged lipid bilayer vesicles (liposomes) using a dye-leakage assay. Our findings show that Au ENMs having both positive and negative surface charge induce leakage that reaches a steady state after several hours. Positively charged particles with identical surface functionality and different core compositions show similar leakage effects and result in faster and greater leakage than negatively charged particles, which suggests that surface functionality, not particle core composition, is a critical factor in determining the interaction between ENMs and lipid bilayers. The results suggest that particles permanently adsorb to bilayers and that only one positively charged particle is required to disrupt a liposome and trigger the leakage of its entire contents in contrast to mellitin molecules, the most widely studied membrane lytic peptide, which requires hundred of molecules to generate leakage.

Topics: Cell Membrane; Engineering; Gold; Kinetics; Melitten; Nanoparticles; Particle Size; Phosphatidylcholines; Surface Properties; Titanium; Unilamellar Liposomes

We describe, in a system whose uniqueness is that the presence of pores allows the volume to vary as budding proceeds, how phase separation on the surface of spheres extrudes material in the process called "budding". The system is giant phospholipid vesicles (GUVs) containing phase-separated regions of DOPC (soft, liquid) and DPPC (stiff, gel), with cholesterol and without it. Budding is triggered by adding the cationic pore-forming peptide, melittin. Without cholesterol, fluorescence experiments show that melittin selectively binds to the liquid domains, inducing them to form mainly exocytotic monodisperse smaller vesicle buds of this same material, causing the parent GUV to shrink. The effect of cholesterol is to produce just a few large buds following domain coalescence, rather than numerous smaller monodisperse ones. Line tension is experimentally shown to be essential for budding in this multicomponent membrane.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cell Membrane; Cholesterol; Lipid Bilayers; Melitten; Peptides; Phosphatidylcholines; Phospholipids; Porosity; Unilamellar Liposomes; Water

The lipid matrix, or the lipid bilayer, of cell membranes is a natural binding site for amphipathic molecules, including antimicrobial peptides, pore-forming proteins, and many drugs. The unique property of pore-forming antimicrobial peptides is that they exhibit a threshold concentration (called the lethal concentration or the minimum inhibitory concentration) for activity, below which no effect is seen. Without this property, antimicrobial peptides would not be effective self-defense weapons, because they would have harmed all cells at any concentration. The question is what gives rise to this unique property? This study provides a free energy description for the origin of a threshold concentration. The same free energy applied differently also explains the binding of drugs that shows no threshold concentrations. The idea is compared with theories of micellar solutions that require a large oligomer size (n 15) to achieve a threshold concentration. The elasticity of lipid bilayers makes the phenomena in membranes different. The majority of antimicrobial peptides have a large negative binding energy to the bilayer interface, but the binding causes an expansion in the membrane area, or equivalently a thinning in the membrane thickness. This elastic energy of membrane thinning elevates the energy level of interfacial binding with the peptide concentration, hence gives rise to a threshold concentration for forming pores containing as few as four peptides.

Topics: Alamethicin; Algorithms; Animals; Antimicrobial Cationic Peptides; Bees; Curcumin; Elasticity; Lipid Bilayers; Melitten; Models, Molecular; Phosphatidylcholines; Thermodynamics

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.

Topics: Animals; Bee Venoms; Bees; Cell Membrane; Circular Dichroism; Dose-Response Relationship, Drug; Fluoresceins; Lipids; Liposomes; Melitten; Molecular Conformation; Phosphatidylcholines; Protein Structure, Tertiary; Surface Properties

Our understanding of how antimicrobial and cell-penetrating peptides exert their action at cell membranes would benefit greatly from direct visualization of their modes of action and possible targets within the cell membrane. We previously described how the cationic antimicrobial peptide, indolicidin, interacted with mixed zwitterionic planar lipid bilayers as a function of both peptide concentration and lipid composition [Shaw, J.E. et al., 2006. J. Struct. Biol. 154 (1), 42-58]. In the present report, in situ atomic force microscopy was used to characterize the interactions between three families of cationic peptides: (1) tryptophan-rich antimicrobial peptides--indolicidin and two of its analogues, (2) an amphiphilic alpha-helical membranolytic peptide--melittin, and (3) an arginine-rich cell-penetrating peptide--Tat with phase-separated planar bilayers containing 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) or DOPC/N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM)/cholesterol. We found that these cationic peptides all induced remodelling of the model membranes in a concentration, and family-dependent manner. At low peptide concentration, these cationic peptides, despite their different biological roles, all appeared to reduce the interfacial line tension at the domain boundary between the liquid-ordered and liquid-disordered domains. Only at high peptide concentration was the membrane remodelling induced by these peptides morphologically distinct among the three families. While the transformation caused by indolicidin and its analogues were structurally similar, the concentration required to initiate the transformation was strongly dependent on the hydrophobicity of the peptide. Our use of lipid compositions with no net charge minimized the electrostatic interactions between the cationic peptides and the model supported bilayers. These results suggest that peptides within the same functional family have a common mechanism of action, and that membrane insertion of short cationic peptides at low peptide concentration may also alter membrane structure through a common mechanism regardless of the peptide's origin.

Topics: Antimicrobial Cationic Peptides; Cholesterol; Dimyristoylphosphatidylcholine; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Liposomes; Melitten; Microscopy, Atomic Force; Phosphatidylcholines; Sphingomyelins

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. The organization of membrane-bound melittin has earlier been shown to be dependent on the physical state and composition of membranes. In this study, we covalently labeled the N-terminal (Gly-1) and Lys-7 of melittin with an environment-sensitive fluorescent probe, the NBD group, to monitor the influence of negatively charged lipids and cholesterol on the organization and dynamics of membrane-bound melittin. Our results show that the NBD group of melittin labeled at its N-terminal end does not exhibit red edge excitation shift in DOPC and DOPC/DOPG membranes, whereas the NBD group of melittin labeled at Lys-7 exhibits REES of approximately 8 nm. This could be attributed to difference in membrane microenvironment experienced by the NBD groups in these analogs. Interestingly, the membrane environment of the NBD groups is sensitive to the presence of cholesterol, which is supported by time-resolved fluorescence measurements. Importantly, the orientation of melittin is found to be parallel to the membrane surface as determined by membrane penetration depth analysis using the parallax method in all cases. Our results constitute the first report to our knowledge describing the orientation of melittin in cholesterol-containing membranes. These results assume significance in the overall context of the role of membrane lipids in the orientation and function of membrane proteins and peptides.

Topics: Amino Acid Sequence; Animals; Bees; Cholesterol; Fluorescence Polarization; Fluorescent Dyes; Hemolysis; In Vitro Techniques; Lipid Bilayers; Melitten; Molecular Conformation; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Rats; Rats, Wistar

We have recently shown that current molecular dynamics (MD) atomic force fields are not yet able to produce lipid bilayer structures that agree with experimentally-determined structures within experimental errors. Because of the many advantages offered by experimentally validated simulations, we have developed a novel restraint method for membrane MD simulations that uses experimental diffraction data. The restraints, introduced into the MD force field, act upon specified groups of atoms to restrain their mean positions and widths to values determined experimentally. The method was first tested using a simple liquid argon system, and then applied to a neat dioleoylphosphatidylcholine (DOPC) bilayer at 66% relative humidity and to the same bilayer containing the peptide melittin. Application of experiment-based restraints to the transbilayer double-bond and water distributions of neat DOPC bilayers led to distributions that agreed with the experimental values. Based upon the experimental structure, the restraints improved the simulated structure in some regions while introducing larger differences in others, as might be expected from imperfect force fields. For the DOPC-melittin system, the experimental transbilayer distribution of melittin was used as a restraint. The addition of the peptide caused perturbations of the simulated bilayer structure, but which were larger than observed experimentally. The melittin distribution of the simulation could be fit accurately to a Gaussian with parameters close to the observed ones, indicating that the restraints can be used to produce an ensemble of membrane-bound peptide conformations that are consistent with experiments. Such ensembles pave the way for understanding peptide-bilayer interactions at the atomic level.

Topics: Birefringence; Computer Simulation; Lipid Bilayers; Melitten; Membrane Proteins; Models, Chemical; Models, Molecular; Peptides; Phosphatidylcholines; Stress, Mechanical

An optical technique, dual-polarization interferometry, has been used to examine lipid structures at the solid/liquid interface. Changes in the lipid structures, in real time, were examined as a consequence of challenging them with a peptide (melittin) that is known to induce liposome rupture. This work suggests that it should be possible to obtain a better understanding of the detail of the melittin rupture process.

Topics: Fluorescence Polarization; Lipid Bilayers; Liposomes; Melitten; Phosphatidylcholines

Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.

Topics: Alamethicin; Animals; Anti-Bacterial Agents; Circular Dichroism; Ion Channels; Lipid Bilayers; Melitten; Membranes, Artificial; Models, Chemical; Phosphatidylcholines; Protein Binding; Spectroscopy, Fourier Transform Infrared; Thermodynamics; X-Ray Diffraction

We have monitored the organization and dynamics of the hemolytic peptide melittin in membranes containing cholesterol by utilizing the intrinsic fluorescence properties of its functionally important sole tryptophan residue and circular dichroism spectroscopy. The significance of this study is based on the fact that the natural target for melittin is the erythrocyte membrane, which contains high amounts of cholesterol. Our results show that the presence of cholesterol inhibits melittin-induced leakage of lipid vesicles and the extent of inhibition appears to be dependent on the concentration of membrane cholesterol. The presence of cholesterol is also shown to reduce binding of melittin to membranes. Our results show that fluorescence parameters such as intensity, emission maximum, and lifetime of membrane-bound melittin indicate a change in polarity in the immediate vicinity of the tryptophan residue probably due to increased water penetration in presence of cholesterol. This is supported by results from fluorescence quenching experiments using acrylamide as the quencher. Membrane penetration depth analysis by the parallax method shows that the melittin tryptophan is localized at a relatively shallow depth in membranes containing cholesterol. Analysis of energy transfer results using melittin tryptophan (donor) and dehydroergosterol (acceptor) indicates that dehydroergosterol is not randomly distributed and is preferentially localized around the tryptophan residue of membrane-bound melittin, even at the low concentrations used. Taken together, our results are relevant in understanding the interaction of melittin with membranes in general, and with cholesterol-containing membranes in particular, with possible relevance to its interaction with the erythrocyte membrane.

Topics: Binding Sites; Cholesterol; Kinetics; Liposomes; Macromolecular Substances; Melitten; Membrane Proteins; Membranes, Artificial; Phosphatidylcholines; Protein Binding; Spectrometry, Fluorescence; Tryptophan

Electric fields play an important role in the physiological function of macromolecules. Much is known about the role that electric fields play in biological systems, but membrane molecule structure and orientation induced by electric fields remain essentially unknown. In this paper, we present a polarized attenuated total reflection (ATR) experiment we designed to study the effect of electric fields on membrane molecule structure and orientation by Fourier-transform infrared (FTIR) spectroscopy. Two germanium crystals used as the internal reflection element for ATR-FTIR experiments were coated with a thin layer of polystyrene as insulator and used as electrodes to apply an electric field on an oriented stack of membranes made of dioleylphosphatidylcholine (DOPC) and melittin. This experimental set up allowed us for the first time to show fully reversible orientational changes in the lipid headgroups specifically induced by the electric potential difference.

Topics: Biophysical Phenomena; Biophysics; Electrochemistry; In Vitro Techniques; Liposomes; Melitten; Membrane Lipids; Membrane Potentials; Phosphatidylcholines; Spectroscopy, Fourier Transform Infrared

Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin's exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

Topics: Animals; Biophysical Phenomena; Biophysics; Circular Dichroism; Dimerization; In Vitro Techniques; Lipid Bilayers; Melitten; Membrane Lipids; Membrane Proteins; Models, Molecular; Phosphatidylcholines; Protein Structure, Quaternary; Protein Structure, Secondary; X-Ray Diffraction

The effects of covalent dimerisation of melittin by disulphide formation in cysteine-substitution analogues, (melittin K23C)2 and (melittin K23Q,Q25C)2, on the kinetics of pore formation in phosphatidylcholine small unilamellar vesicles was measured under low ionic strength conditions. The initial rate of melittin-induced pore formation increased with the square of the peptide concentration, whereas both disulphide-dimerised melittin analogues showed a first-order dependence of pore formation rates on peptide concentration. These results indicate that peptide dimerisation is rate-limiting for pore formation under these conditions. A model for a generalised bilayer perturbation resulting from the self-association of a pair of peptide helices at the membrane surface is proposed which may have implications for a number of biological processes that involve the interaction of helical polypeptides with membranes.

Topics: Animals; Dimerization; Dimyristoylphosphatidylcholine; Disulfides; In Vitro Techniques; Lipid Bilayers; Melitten; Membrane Proteins; Models, Molecular; Phosphatidylcholines; Protein Conformation; Protein Structure, Secondary; Surface Properties

We have investigated, both experimentally and theoretically, the efflux of carboxyfluorescein (a self-quenching fluorescent dye) from vesicles of different sizes and lipid species (POPC, DOPC) after having added the bee venom peptide melittin. This comprises quantitative analyses regarding the extent of lipid-associated peptide, the mode as well as the temporal progress of dye release and the possible leakage mechanism. Our results indicate a graded efflux characterized by a single-pore retention factor reflecting the formation of pores whose lifetimes are rather small (millisecond range). The observed fluorescence signal arising from the dequenching of effluent dye has been converted to the number of pore openings over the course of time. All the resulting curves exhibit a pronounced slowing down of the pore formation rate revealing two distinct relaxation steps at about 20 and 200 s, respectively, being largely independent of vesicle type and peptide to lipid ratio. The pore formation rate itself increases in proportion to the amount of membrane bound peptide. We give a quantitative account of our experimental findings based on a novel reaction scheme applicable to any of our various liposome systems. It implies that the pore formation rate is controlled by a passage through two intermediate monomeric peptide states. These states are thought to become well populated in the initial stage of lipid bilayer perturbation, but would practically die out after some time owing to a restabilization of the membrane system.

Topics: Binding Sites; Energy Transfer; Fluoresceins; Fluorescent Dyes; In Vitro Techniques; Kinetics; Liposomes; Melitten; Phosphatidylcholines; Spectrometry, Fluorescence

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. Since the association of the peptide in the membrane is linked with its physiological effects, a detailed understanding of the interaction of melittin with membranes is crucial. We have investigated the interaction of melittin with membranes of varying surface charge in the context of recent studies which show that the presence of negatively charged lipids in the membrane inhibits membrane lysis by melittin. The sole tryptophan residue in melittin has previously been shown to be critical for its hemolytic activity. The organization and dynamics of the tryptophan residue thus become important to understand the peptide activity in membranes of different charge types. Wavelength-selective fluorescence was utilized to monitor the tryptophan environment of membrane-bound melittin. Melittin exhibits a red edge excitation shift (REES) of 5 nm when bound to zwitterionic membranes while in negatively charged membranes, the magnitude of REES is reduced to 2-3 nm. Further, wavelength dependence of fluorescence polarization and near-UV circular dichroism spectra reveal characteristic differences in the tryptophan environment for melittin bound to zwitterionic and anionic membranes. These studies are supported by time-resolved fluorescence measurements of membrane-bound melittin. Tryptophan penetration depths for melittin bound to zwitterionic and anionic membranes were analyzed by the parallax method [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45] utilizing differential fluorescence quenching obtained with phospholipids spin-labeled at two different depths. Our results provide further insight into molecular details of membrane lysis by melittin and the modulation of lytic activity by negatively charged lipids.

Topics: Animals; Bees; Circular Dichroism; Dimyristoylphosphatidylcholine; Kinetics; Lipid Bilayers; Melitten; Models, Molecular; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids; Protein Conformation; Spectrometry, Fluorescence; Structure-Activity Relationship; Tryptophan

We investigated the interaction of the peptide melittin with differently sized vesicles consisting of various lipid compositions. This system was characterized by dynamic light scattering to estimate the size of vesicles. For SUV we obtained a radius of 12 nm, for LUV 53 nm. The pore forming process of melittin in vesicles was investigated by efflux of encapsulated fluorescent dyes at a self-quenching concentration. The influence of the following parameters on efflux and pore formation was estimated: lipid composition (POPC and DOPC), vesicle size (SUV and LUV) and size of the encapsulated dye (carboxyfluorescein and FITC-dextran). We found that under similar conditions vesicles of DOPC give always less leakage than vesicles of POPC independent of the fluorescent dye. For SUV and LUV we have obtained a different leakage behaviour at identical surface concentrations of melittin (if the same partition coefficient is assumed). From efflux measurements with different dyes we concluded that 6-20 molecules of melittin are necessary to form a pore. The possibility that not pore formation but fusion is the mechanism of melittin induced efflux was disproved by fusion experiments using a resonance energy transfer assay.

Topics: Amino Acid Sequence; Cell Membrane; Dextrans; Fluorescein-5-isothiocyanate; Fluoresceins; Light; Liposomes; Melitten; Membrane Fusion; Membrane Lipids; Membranes, Artificial; Molecular Sequence Data; Phosphatidylcholines; Scattering, Radiation

To determine the underlying basis for the sensitivity of peripheral peptides to lipid packing, we monitored the change in association of melittin to different membranes under hydrostatic pressure by fluorescence polarization and by fluorescence intensity in the presence of aqueous quenchers. Association to lysophosphatidylcholine micelles or to membranes composed of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or dioleoylphosphatidylcholine was found to be stable from 1 to 2000 atm. Similar results were obtained using multilamellar vesicles, small unilamellar vesicles, or large unilamellar vesicles. Thus, the increase in lipid chain packing induced by pressure does not alter the association of bound complexes. This result indicates similar compressibilities of the peptide and the head group binding region. Increasing the ionic strength to increase the charge of the free peptide also resulted in a pressure-insensitive complex showing that the hydration does not change upon binding. This conclusion is substantiated by a lack of van't Hoff delta H to dioleoylphosphatidylcholine large unilamellar vesicles. To gain a more molecular picture of these associations, the rotational properties of the tryptophan side chain of bound melittin as a function of lipid packing was also studied. These data indicate subtle differences in peptide orientation in different lipids.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Dimyristoylphosphatidylcholine; Kinetics; Liposomes; Melitten; Osmolar Concentration; Phosphatidylcholines; Pressure; Protein Binding; Spectrometry, Fluorescence; Structure-Activity Relationship; Thermodynamics

Melittin-induced conductance was measured on planar bilayers made from dioleoylphosphatidylcholine. Upon application of a fixed voltage, the current response was monophasic and remained so even after prolonged observation times. The conductance of melittin-doped bilayers increased exponentially with voltage. In addition, an ohmic contribution appeared after some current had passed. The voltage-dependent conductance increased e-fold every 22 mV and was proportional to the fourth power of the aqueous monomeric peptide concentration, for all salt concentrations investigated (0.4-1.8 M NaCl). Discrete conductance steps could be resolved at all these salt concentrations. The amplitudes of these steps were highly variable. In each experiment, conductance was initially only observed for potentials which were positive on the side of peptide addition. As more and more current passed across the bilayer, the current-voltage curves became symmetric. The system needed some time to reach stationary current-voltage characteristics: about 50 min at pH 7 but only about 15 min at pH 8, suggesting involvement of the N-terminus (pK around 7.5) of melittin in the slow formation of a 'prepore'.

Topics: Amino Acid Sequence; Electric Conductivity; Lipid Bilayers; Melitten; Models, Biological; Molecular Sequence Data; Osmolar Concentration; Phosphatidylcholines; Potentiometry

Topics: Bee Venoms; Cholesterol; Electric Conductivity; Lipid Bilayers; Melitten; Membrane Potentials; Phosphatidylcholines; Pronase; Serum Albumin, Bovine; Spectrophotometry; Trypsin; Valinomycin