Peptide inhibitors of enveloped virus infection inhibit phospholipid vesicle fusion and Sendai virus fusion with phospholipid vesicles

Small hydrophobic peptides that are capable of inhibiting Sendai virus infection of cells (Richardson, C. D., Scheid, A., and Choppin, P. W. (1980) Virology 105, 205-222) are also capable of inhibiting membrane fusion in a pure lipid vesicle system. Large unilamellar vesicles of N-methyl dioleoylphosphatidylethanolamine containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid and/or p-xylene bis (pyridinium bromide) were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) fluorescence assay. Sendai virus fusion with lipid vesicles was measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride, a lipid mixing assay for fusion. The efficiency with which the peptides carbobenzoxy-D-Phe-L-PheGly, carbobenzoxy-L-Phe-L-Tyr, and carbobenz-oxy-Gly-L-Phe inhibit fusion of N-methyl dioleoyl-phosphatidylethanolamine large unilamellar vesicles directly paralleled their previously known effectiveness in blocking virus infectivity of cultured cells. In addition, above a certain concentration threshold, the inhibitory peptides decreased the initial rate of leakage from lipid vesicles. The inhibition by these peptides of virus-vesicle fusion followed the same order of potency as for vesicle-vesicle fusion. The observation of the same relative potency of these peptides toward inhibition of virus-cell infection, and virus-vesicle and vesicle-vesicle membrane fusion suggested that these peptides inhibited virus-cell infection by inhibiting the ability of the virus to fuse with the cell. Furthermore, these results suggest that the mechanism of inhibition of all three fusion events may have steps in common.     

Fusogenic activity of δ-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin δ-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of δ-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Poly(ethylene glycol)-induced lipid mixing but not fusion between synthetic phosphatidylcholine large unilamellar vesicles

We have examined the effect of poly(ethylene glycol) (PEG) on stable large unilamellar vesicles formed by a rapid extrusion technique and composed of pure synthetic phosphatidylcholines. The lipid systems studied were the saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the monounsaturated 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). PEG at all concentrations (3.8-40 wt %) induced lipid mixing between large vesicles composed of these phosphatidylcholines. Extensive leakage of internal contents also occurred at high PEG concentrations. However, in contrast to our previous report [Parente, R. A., & Lentz, B. R. (1986) Biochemistry 25, 6678], we could detect no mixing of internal contents indicative of fusion. This discrepancy is due to environmental factors that affect the behavior of 8-amino-naphthalene-1,3,6-trisulfonic acid (ANTS), the fluorophore used in the assay for contents mixing and leakage [McIntyre, Parks, Massenburg, & Lentz (1991) (submitted)]. In agreement with the results of the fusion assay, quasielastic light-scattering measurements revealed no increase in vesicle size following treatment with PEG. These results emphasize the importance of using assays for both membrane mixing and contents mixing to demonstrate fusion, since significant lipid mixing occurred in the absence of fusion. We conclude that large vesicles composed of pure phosphatidylcholine do not fuse in the presence of even high concentrations of PEG. However, DOPC vesicles containing a small amount of an amphipathic "impurity" have been shown to fuse in the presence of PEG at 23 degrees C. These results are discussed in terms of their implications for the mechanism of PEG-induced membrane fusion.     

Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues

Egg sphingomyelin vesicles were used to assay aggregation/fusion activities of proteins from Taiwan (Naja naja atra) venom to avoid the problem of phospholipase A2 contamination during protein purification. It led to the identification of a new cardiotoxin (CTX) analogue protein (CTX V) with major aggregation/fusion, but few hemolysis, activities. On the contrary, cardiotoxin (CTX III) induced significant hemolysis of human red blood cells but exhibited few aggregation/fusion activities. To study the structure/activity relationship of these CTX-induced processes, the amino acid sequence of CTX V was determined and its aggregation/fusion activity was compared with that of CTX III by transmission electron microscopy, quasielastic laser light scattering, differential scanning calorimetry, and fluorescence spectroscopy. The results show that the CTX-induced fusion process at temperatures slightly above that of the gel to liquid-crystalline phase transition of sphingomyelin vesicles can ultimately convert small sonicated vesicles into large fused vesicles with sizes of 1-2 microns. The abilities of CTX V to induce the leakage of sphingomyelin vesicles content and to cause the fusion of vesicles are approximately 10-fold higher than those of CTX III. Based on the CTX structures determined in the present and other studies, it is suggested that the amino acid residue X within the well conserved sequence of -Cys-Pro-X-Gly-Lys-Gln-Leu-Cys- plays a role in the interaction of CTX with lipid molecules. The lipid phase transition could further enhance the protein-lipid interaction in the process leading to the fusion of vesicles.     

Spontaneous vesicularization of myelin lipids is counteracted by myelin basic protein

Hand-vortexed dispersions of several lipids (cerebrosides, sulfatides, PC, PE, PS and sphingomyelin), mixed in the ratios found for these categories of lipids in myelin, exhibit 31P-NMR spectra which have contributions from both isotropic and lamellar resonances. Investigation of this system by freeze-fracture electron microscopy and X-ray diffraction revealed that this lipid mixture has spontaneously formed small unilamellar vesicles (SUVs) (diam. approximately 400 A) and large highly convoluted unilamellar vesicles (LUVs) (diam. approximately 1000 A), the latter possibly resulting from aggregation and fusion of the SUV structures. This vesicularization of the myelin lipids was reversed by the addition of myelin basic protein: only large multilamellar aggregates were formed in the presence of protein, as shown by all three experimental methods. Although no rigorous physical-chemical explanation for these phenomena is yet available, the possibility is suggested that the high concentration of cerebrosides and/or phosphatidylethanolamine in this particular mixture of myelin lipids play pivotal roles in the formation of these unusual vesicles. Spontaneous vesicularization of myelin lipids is discussed as a potential pathway toward destabilization of the myelin sheath.     

Electron microscopic investigation of the morphology and calcium-induced fusion of lipid vesicles with an oligomerised inner leaflet

The lipid head groups in the inner leaflet of unilamellar bilayer vesicles of the synthetic lipids DHPBNS and DDPBNS can be selectively oligomerised. Earlier studies have established that these vesicles fuse much slower and less extensively upon oligomerisation of the lipid head groups. The morphology and calcium-induced fusion of vesicles of DHPBNS and DDPBNS were investigated using cryo-electron microscopy. DHPBNS vesicles are not spherical but flattened, ellipsoidal structures. Upon addition of CaCl(2), DHPBNS vesicles with an oligomerised inner leaflet were occasionally observed in an arrested hemifused state. However, the evidence for hemifusion is not equivocal due to potential artefacts of sample preparation. DDPBNS vesicles show the expected spherical morphology. Upon addition of excess CaCl(2), DDPBNS vesicles fuse into dense aggregates that show a regular spacing corresponding to the bilayer width. Upon addition of EDTA, the aggregates readily disperse into large unilamellar vesicles. At low concentration of calcium ion, DDPBNS vesicles with an oligomerised inner leaflet form small multilamellar aggregates, in which a spacing corresponding to the bilayer width appears. Addition of excess EDTA results in slow dispersal of the Ca2+-lipid aggregates into a heterogeneous mixture of bilamellar, spherical vesicles and networks of thread-like vesicles. These lipid bilayer rearrangements are discussed within the context of shape transformations and fusion of lipid membranes.     

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.     

Protein machines and lipid assemblies: current views of cell membrane fusion

Protein machines and lipid bilayers both play central roles in cell membrane fusion, a process crucial to life. Recent results provide clues to how both components function in fusion. Recent observations suggest a common mechanism by which very different fusion machines (from lipid-enveloped viruses and synaptic vesicles) may function to produce compartment-joining pores. This mechanism presumes that fusion proteins act as machines that use stored conformational energy to assemble closely juxtaposed lipid bilayers, bend these to form fusion-competent structures, stabilize unfavorable lipid structures and destabilize a committed intermediate to drive fusion pore formation.     

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane interactions and membrane fusion.

Results of a kinetic model of thermotropic L alpha----HII phase transitions are used to predict the types and order-of-magnitude rates of interactions between unilamellar vesicles that can occur by intermediates in the L alpha----HII phase transition. These interactions are: outer monolayer lipid exchange between vesicles; vesicle leakage subsequent to aggregation; and (only in systems with ratios of L alpha and HII phase structural dimensions in a certain range or with unusually large bilayer lateral compressibilities) vesicle fusion with retention of contents. It was previously proposed that inverted micellar structures mediate membrane fusion. These inverted micellar structures are thought to form in all systems with such transitions. However, I show that membrane fusion probably occurs via structures that form from these inverted micellar intermediates, and that fusion should occur in only a sub-set of lipid systems that can adopt the HII phase. For single-component phosphatidylethanolamine (PE) systems with thermotropic L alpha----HII transitions, lipid exchange should be observed starting at temperatures several degrees below TH and at all higher temperatures, where TH is the L alpha----HII transition temperature. At temperatures above TH, the HII phase forms between apposed vesicles, and eventually ruptures them (leakage). In most single-component PE systems, fusion via L alpha----HII transition intermediates should not occur. This is the behavior observed by Bentz, Ellens, Lai, Szoka, et al. in PE vesicle systems. Fusion is likely to occur under circumstances in which multilamellar samples of lipid form the so-called "inverted cubic" or "isotropic" phase. This is as observed in the mono-methyl DOPE system (Ellens, H., J. Bentz, and F. C. Szoka. 1986. Fusion of phosphatidylethanolamine containing liposomes and the mechanism of the L alpha-HII phase transition. Biochemistry. In press.) In lipid systems with L alpha----HII transitions driven by cation binding (e.g., Ca2+-cardiolipin), fusion should be more frequent than in thermotropic systems.     

Temperature-induced fusion of small unilamellar vesicles formed from saturated long-chain lecithins and diheptanoylphosphatidylcholine

Small unilamellar vesicles which form when gel-state long-chain phosphatidylcholines are mixed with micellar short-chain lecithins undergo an increase in size as the long-chain species melts to its liquid-crystalline form. Analysis of the vesicle population with quasi-elastic light scattering shows that the particle size increases from 90-A radius to greater than 5000-A radius. Resonance energy transfer experiments show total mixing of lipid probes with unlabeled vesicles only when the Tm of the long-chain phosphatidylcholine is exceeded. This implies that the large size change represents a fusion process. Aqueous compartments are also mixed during this transition. 31P NMR analysis of the vesicle mixtures above the phase transition shows a great degree of heterogeneity with large unilamellar particles coexisting with oligo- and multilamellar structures. Upon cooling the vesicles below the Tm, the original size distribution (e.g., small unilamellar vesicles) is obtained, as monitored by both quasi-elastic light scattering and 31P NMR spectroscopy. This temperature-induced fusion of unilamellar vesicles is concentration dependent and can be abolished at lower total phospholipid concentrations. It occurs over a wide range of long-chain to short-chain ratios and occurs with 1-palmitoyl-2-stearoylphosphatidylcholine and dimyristoylphosphatidylcholine as well. Characterization of this fusion event is used to understand the anomalous kinetics of water-soluble phospholipases toward these unusual vesicles.     

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

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

The effects of α-synuclein on phospholipid vesicle integrity: a study using 31P NMR and electron microscopy

Associations between the 140 amino acid protein α-synuclein (asyn) and presynaptic vesicles may play a role in maintaining synaptic plasticity and neurotransmitter release. These physiological processes may involve disruption and fusion of vesicles, arising from interactions between specific regions of asyn, including the highly basic N-terminal domain, and the surface of vesicles. This work investigates whether asyn affects the integrity of model unilamellar vesicles of varying size and phospholipid composition, by monitoring paramagnetic Mn²⁺-induced broadening of peaks in the ³¹P nuclear magnetic resonance spectrum of the lipid head groups. It is shown that asyn increases the permeability to Mn²⁺ of both large (200nm diameter) and small (50nm diameter) vesicles composed of zwitterionic phosphatidylcholine and anionic phosphatidylglycerol at protein/lipid molar ratios as low as 1:2000. Further experiments on peptides corresponding to sequences in the N-terminal (10–48), C-terminal (120–140) and central hydrophobic (71–82) regions of asyn suggest that single regions of the protein are capable of permeabilizing the vesicles to varying extents. Electron micrographs of the vesicles after addition of asyn indicate that the enhanced permeability is coupled to large-scale disruption or fusion of the vesicles. These results indicate that asyn is able to permeabilize phospholipid vesicles at low relative concentrations, dependent upon the properties of the vesicles. This could have implications for asyn playing a role in vesicle synthesis, maintenance and fusion within synapses.     

Bilayer curvature and certain amphipaths promote poly(ethylene glycol)-induced fusion of dipalmitoylphosphatidylcholine unilamellar vesicles.

Unilamellar vesicles of varying and reasonably uniform size were prepared from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) by the extrusion procedure and sonication. Quasi-elastic light scattering was used to show that different vesicle preparations had mean (Z-averaged) diameters of 1340, 900, 770, 630, and 358 A (sonicated). Bilayer-phase behavior as detected by differential scanning calorimetry was consistent with the existence of essentially uniform vesicle populations of different sizes. The response of these different vesicles to treatment with poly(ethylene glycol) (PEG) was monitored using fluorescence assays for lipid transfer, contents leakage, and contents mixing, as well as quasi-elastic light scattering. No fusion, as judged by vesicle contents mixing and change in vesicle size, was detected for vesicles of diameter greater than 770 A. The diameters of smaller vesicles increased dramatically when treated with high concentrations of PEG, although mixing of their contents could not be detected both because of their small trapped volumes and because of the extensive leakage induced in small vesicles by high concentrations of PEG. Lipid transfer was detected between vesicles of all sizes. We conclude the high bilayer curvature does encourage fusion of closely juxtaposed membrane bilayers but that highly curved vesicles appear also to rupture and form larger structures when diluted from high PEG concentration, a process that can be confused with fusion. Despite the failure of PEG to induce fusion of large, uncurved vesicles composed of a single phosphatidylcholine, these vesicles can be induced to fuse when they contain small amounts of certain amphiphathic compounds thought to play a role in cellular fusion processes. Thus, vesicles which contained 0.5 mol % L-alpha-lysopalmitoylphosphatidylcholine, 5 mol % platelet activating factor, or 0.5 mol % palmitic acid fused in the presence of 30%, 25%, and 20% (w/w) PEG, respectively. However, vesicles containing 1,2-dipalmitoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, 1-oleoyl-2-acetyl-sn-glycerol, or monooleoyl-rac-glycerol at surface concentrations up to 5 mol % did not fuse in the presence or absence of PEG. There was no correlation between the abilities of these amphipaths to induce phase separation or nonlamellar phases and their abilities to support fusion of pure DPPC unilamellar vesicles in the presence of high concentrations of PEG. The results are discussed in terms of the type of disrupted lipid packing that could be expected to favor PEG-mediated fusion.     

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca²⁺-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1–2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal H_II phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca²⁺, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca²⁺ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.     

Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions

Summary Phospholipid vesicles (liposomes) were subjected to dehydration-hydration cycles in the presence of 6-carboxyfluorescein or salmon sperm DNA. We found that the vesicles fused into multilamellar structures during dehydration with solutes trapped between the lamellae. Upon rehydration the lamellae swelled and formed large vesicular structures containing solute. This model can be used to study encapsulation of macromolecules by lipid membranes to form protocellular structures under prebiotic conditions.     

The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean (Vicia faba) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.     

1,2-Dioleoylglycerol promotes calcium-induced fusion in phospholipid vesicles.

The effect of 1,2-dioleoyglycerol (1,2-DOG) on the promotion of Ca(2+)-induced fusion of phosphatidylserine/phosphatidylcholine (PS/PC) vesicles was studied. 1,2-DOG is able to induce the mixing of membrane lipids at concentrations of 10 mol% without mixing of vesicular contents. At concentrations of 20 mol% or higher, 1,2-DOG promotes fusion, lipid and content mixing, of LUV composed of an equimolar mixture of PS and PC, which otherwise are unable to fuse in the presence of Ca2+. Fusion was demonstrated by fluorescence assays monitoring mixing of aqueous vesicular contents and mixing of membrane lipids. Studies by Fourier transform infrared spectroscopy provided evidence for a fusion mechanism different to that of Ca(2+)-induced fusion of pure PS vesicles. Final equilibrium structures were characterized by 31P-NMR and freeze-fracture electron microscopy. Ca(2+)-induced fusion of 1,2-DOG containing vesicles is accompanied by the formation of isotropic structures which are shown to correspond to structures with lipidic particle morphology. The possible fusion mechanisms and implications are discussed.     

Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.     

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.