Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles

We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar vesicles (diameter approximately 100 nm) composed of phosphatidylcholine and phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large unilamellar vesicles are significantly larger than the peptide, the correlation times for the free and bound peptide could be distinguished using single color autocorrelation measurements. The molar partition coefficients calculated from the FCS measurements were comparable to those obtained from binding measurements of radioactively labeled MARCKS(151-175) using a centrifugation technique. Moreover, FCS can measure binding of peptides present at very low concentrations (1-10 nmolar), which is difficult or impossible with most other techniques. Our data indicate FCS can be an accurate and valuable tool for studying the interaction of peptides and proteins with lipid membranes.     

The effect of a membrane potential on the interaction of mastoparan X, a mitochondrial presequence, and several regulatory peptides with phospholipid vesicles.

Recently the pH gradient evoked by a K+ diffusion potential was shown to translocate a synthetic monobasic amphipathic hexapeptide across the bilayer of lipid vesicles (De Kroon, A.I.P.M., Vogt, B., Van 't Hof, R., De Kruijff, B. and De Gier, J. (1991) Biophys. J. 60, in press). Here this observation is extended by studying the effect of a membrane potential on a set of bioactive peptides. The panel of peptides comprises the toxin mastoparan X, a tryptophan-containing analogue of the presequence of the mitochondrial protein cytochrome oxidase subunit IV (preCoxIV(1-25)W18), and the regulatory peptides ACTH(1-24), alpha-MSH, ACTH(1-10), dynorphin A, bombesin, and LHRH. The interaction of these peptides with phospholipid vesicles has been measured using the intrinsic tryptophan residue as fluorescent probe. In the absence of a K+ diffusion potential only mastoparan X and the presequence show considerable binding to vesicles consisting of phosphatidylcholine (PC). In contrast, under these conditions all peptides display affinity for vesicles consisting of the acidic phospholipid cardiolipin (CL), the extent of which depends on the net positive charge of the peptide. Application of a K+ diffusion potential to large unilamellar vesicles (LUV) consisting of PC results in a time dependent tryptophan fluorescence increase for mastoparan X, which is accelerated upon incorporating increasing amounts of CL into the LUV. A similar fluorescence increase in response to a K+ diffusion potential was observed for the above model peptide. Yet the mechanism resulting in the fluorescence increase of mastoparan X is completely different from that of the hexapeptide. Binding experiments indicate that a membrane potential-induced enhanced binding of the peptide to the outer surface of the vesicles contributes to the fluorescence increase. PreCoxIV(1-25)W18, dynorphin A, and ACTH(1-24) show fluorescence responses upon applying a membrane potential that are consistent with that of mastoparan X, whereas the other peptides tested do not respond up to a LUV CL content of 50%. The results tentatively suggest that the membrane potential only affects a peptide when it has the ability to adopt a stable membrane bound conformation.     

Fluorescence correlation spectroscopy in biology,chemistry, and medicine

This review describes the method of fluorescence correlation spectroscopy (FCS) and its applications. FCS is used for investigating processes associated with changes in the mobility of molecules and complexes and allows researchers to study aggregation of particles, binding of fluorescent molecules with supramolecular complexes, lipid vesicles, etc. The size of objects under study varies from a few angstroms for dye molecules to hundreds of nanometers for nanoparticles. The described applications of FCS comprise various fields from simple chemical systems of solution/micelle to sophisticated regulations on the level of living cells. Both the methodical bases and the theoretical principles of FCS are simple and available. The present review is concentrated preferentially on FCS applications for studies on artificial and natural membranes. At present, in contrast to the related approach of dynamic light scattering, FCS is poorly known in Russia, although it is widely employed in laboratories of other countries. The goal of this review is to promote the development of FCS in Russia so that this technique could occupy the position it deserves in modern Russian science.     

Pore-forming action of mastoparan peptides on liposomes: a quantitative analysis.

We have investigated the wasp venom peptides mastoparan X and polistes mastoparan regarding their apparent potential to induce pore-like defects in phosphatidylcholine unilamellar vesicles. Based on a fundamental theoretical model, the pore activation and deactivation kinetics have been evaluated from the observed efflux of liposome entrapped carboxyfluorescein in relation to the bound peptide to lipid ratio. We can quantitatively describe our experimental data very well in terms of a specific reaction scheme resulting in only a few short-lived pores. They evidently emerge rapidly from a prepore nucleus being produced by two rate-limiting monomeric states of bound peptide. These peculiar states would be favorably populated in an early stage of bilayer perturbation, but tend to die out in the course of a peptide/lipid restabilization process.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Fluorescence analysis of size distribution and mode of dye release from carboxyfluorescein-loaded vesicles: application to the study of complement-membrane interactions

We wish to report a novel method for visualizing large unilamellar vesicles loaded with a fluorescent dye and for monitoring changes in the size distribution as well as state of aggregation of such dye-loaded liposomes. In addition, we demonstrate that this method can be used to distinguish between all-or-none release of dye and graded release of dye from individual vesicles. Using this technique, we have characterized complement-mediated release of carboxyfluorescein from large unilamellar vesicles and have found that C5-8 complexes mediate a graded release of dye while C5-9 complexes cause an all-or-none release. Furthermore, complement appears to preferentially attack the medium to larger-sized vesicles in our population of large unilamellar vesicles while smaller vesicles appear to be selectively spared.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

The effect of variable liposome brightness on quantifying lipid-protein interactions using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition coefficients, we first show that the variation in liposome brightness caused by this statistical distribution must be considered explicitly in data analysis when the parameter used to establish the partition curves is the fractional instead of the absolute amplitudes associated with the slowest diffusing particles in the system (lipid vesicles), a choice frequently made in FCS partition studies. We further extend the theoretical model describing the membrane partition of a fluorescently-labeled protein by considering the presence of a trace amount of free fluorescent dye (non-binding component) in the system. We show that this situation can account for an apparent maximal binding level lower than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions.     

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

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

Interaction of wheat alpha-thionin with large unilamellar vesicles.

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.     

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Lipid packing determines protein-membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.     

tBid forms a pore in the liposome membrane

We investigated the ability of tBid (truncated form of Bid) to bind and permeabilize the liposomes (large unilamellar vesicles, LUVs) and release fluorescent marker molecules (fluorescein-isothiocyanate-conjugated dextrans, FITC-dextrans) of various molecular diameters (FD-20, FD-70, FD-250S) from LUVs. Obtained data showed that tBid was more efficient in promoting leakage of FITC-dextrans from LUVs composed of cardiolipin and dioleoylphosphatidylcholine (DOPC) than LUVs made of dioleoylphosphatidic acid or dioleoylphosphatidylglycerol and DOPC. The leakage efficiency was reduced with increasing amount of dioleoylphosphatidylethanolamine or dielaidoylphosphatidylethanolamine. Phospholipid monolayer assay and fluorescence quenching measurements revealed that tBid inserted deeply into the hydrophobic acyl chain of acidic phospholipids. Taking into account the tBid three-dimensional structure, we propose that tBid could penetrate into the hydrophobic core of membrane, resulting in the leakage of entrapped content from LUVs via a pore-forming mechanism.     

On the decrease in lateral mobility of phospholipids by sugars

Upon cold and drought stress, sucrose and trehalose protect membrane structures from fusion and leakage. Similarly, these sugars protect membrane proteins from inactivation during dehydration. We studied the interactions between sugars and phospholipid membranes in giant unilamellar vesicles with the fluorescent lipid analog 3,3′-dioctadecyloxacarbocyanine perchlorate incorporated. Using fluorescence correlation spectroscopy, it was found that sucrose decreased the lateral mobility of phospholipids in the fully rehydrated, liquid crystalline membrane more than other sugars did, including trehalose. To describe the nature of the difference in the interaction of phospholipids with sucrose and trehalose, atomistic molecular dynamics studies were performed. Simulations up to 100 ns showed that sucrose interacted with more phospholipid headgroups simultaneously than trehalose, resulting in a larger decrease of the lateral mobility. Using coarse-grained molecular dynamics, we show that this increase in interactions can lead to a relatively large decrease in lateral phospholipid mobility.     

Single GUV method reveals interaction of tea catechin (-)-epigallocatechin gallate with lipid membranes

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration ( approximately 150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 muM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.     

Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes.

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases.     

A fluorescence quenching technique for the measurement of paramagnetic ion concentrations at the membrane/water interface. Intrinsic and X537A-mediated cobalt fluxes across lipid bilayer membranes

We have characterized the quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine by Co2+ in egg phosphatidylcholine (PC) lipid bilayer vesicles. The quenching constant obtained is 59 M-1. We demonstrate one use of this fluorescence quenching technique by measuring intrinsic and X537A-mediated transmembrane Co2+ fluxes in large unilamellar PC vesicles. The intrinsic rate constant for Co2+ flux we measure is 3 X 10(-6) S-1. We confirm that the neutral Co approximately (X537A)2 complex is the main component of the X537A-mediated cobalt flux. Since this method measures the concentration of Co2+ at the site of the fluorophore, it is generally applicable to the measurement of paramagnetic ion concentrations in the region of the membrane/water interface.     

Direct observation of lipid domains in free standing bilayers: from simple to complex lipid mixtures

The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this short presentation, I will address the most relevant findings reported from our laboratory, regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylamino-naphthalene (LAURDAN) under the two-photon excitation fluorescence microscopy will be particularly addressed, especially, the possibility to obtain information about the phase-state of different lipid domains directly from the fluorescent images.     

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.