Penetration and fusion of phospholipid vesicles by lysozyme

The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine ([125I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [125I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.     

Ovalbumin-induced fusion of acidic phospholipid vesicles at low pH

The interactions of ovalbumin (OA) with large unilamellar vesicles (LUV) of phosphatidylserine (PS) and PS/phosphatidylethanolamine (PE) were studied. It was observed that OA induces aggregation, destabilization, and fusion of these LUV composed of acidic phospholipids at low pH levels. The fusion of LUV by OA was monitored by measuring the intermixing of internal aqueous contents of vesicles, by resonance energy transfer assay which follows the mixing of the membrane components, and by thin-sectioning electron microscopy. The pH profile of fusion was found to be similar to the pH-dependent binding of OA to the same phospholipid vesicles. Proteolytic digestion and hydrophobic labeling with dansyl chloride and photoreactive phosphatidylcholine (PC) of the OA-vesicle complex showed that a segment of OA with a molecular weight of approximately 2,500 penetrates the bilayer. The amino acid composition of this segment indicated that it is the 291-322 fragment and not the putative signal sequence.     

Fusion of phospholipid vesicles mediated by cytochrome c

Fusion of phosphatidylserine/phosphatidylethanolamine (1/1) vesicles induced by cytochrome c is studied at a wide range of pH values. A pH profile for the fusion with maximum values at pH 5 and pH 8 is obtained and this is found to be similar to the profile for cytochrome c binding to the vesicles. The binding property of apocytochrome c to the same phospholipid vesicles is found to be about the same as that of the cytochrome c at low ionic strength, but very different at high salt concentrations. No appreciable fusion of vesicles by apocytochrome c is observed. Proteolytic treatment and dansyl chloride labeling of cytochrome c- and apocytochrome c-vesicle complexes show that the C-terminal segments of these proteins with molecular weights of about 3000 and 5000, respectively, penetrate the bilayer. The hydrophobic labeling studies with photoreactive phosphatidylcholine in the bilayer show that segments of both cytochrome c and apocytochrome c go deep into the bilayer.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

Dynamics of proteoliposome formation. Intermediate states during detergent dialysis.

1. The intermediate structures formed during dialysis of mixtures of cholate, phospholipid and cytochrome c oxidase were analysed by gel chromatography and electron microscopy. Measurements of trapped phosphate and the degree of respiratory control were used to assess the integrity of the vesicular structures formed. Protein orientation in the bilayer was monitored by the accessibility of cytochrome c to cytochrome c oxidase. 2. The results indicate that proteoliposome formation by the detergent-dialysis procedure takes place in three distinct stages. In the first stage, cholate/phospholipid and cholate/phospholipid/protein micelles coexist in solution and grow in size as the detergent is slowly removed. At a detergent/phospholipid molar ratio of about 0.2, micelle fusion results in the formation of large bilayer aggregates permeable to both phosphate and cytochrome c. It is at this stage that cytochrome c oxidase is incorporated into the bilayer. In the final stage of dialysis the bilayer sheets fragment into small unilamellar vesicles. 3. The orientation of membrane protein in the final vesicles appears to be determined by the effect of protein conformation on the initial curvature of the bilayer sheets during the fragmentation process.     

Alpha-lactalbumin binding to membranes: evidence for a partially buried protein

The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine was demonstrated by column chromatographic analyses on Sephadex G-200 (10 mM Tris-HCl, pH 7.4, 26-28 degrees C) and by intrinsic fluorescence emission of SUV-bound alpha-LA. It was shown that apo-alpha-LA slowly incorporated into the DMPC vesicle bilayer after equilibrating different mixtures of protein and SUV for several hours. The intrinsic fluorescence properties of the bound apo-alpha-LA were altered only slightly (lambda maxem = 333 nm vs. 337 nm in aqueous solution). The large blue shift in apo-alpha-LA fluorescence in solution induced by monovalent cations, such as Na(I), was almost completely prevented when apo-alpha-LA was membrane bound. Furthermore, the addition of calcium caused a slow conversion from apo-alpha-LA to Ca(II)-alpha-LA by a mechanism consistent with passive diffusion of Ca(II) into the bilayer interior to the (buried) calcium binding site. The release of Ca(II)-alpha-LA from the membrane is discussed with reference to alpha-LA release from the smooth endoplasmic reticulum in vivo.     

Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling

Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions (approximately pH 5) this protein undergoes a conformational change that triggers the exposure of the "fusion peptide", the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane?) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine ([3H]PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed us to investigate both the interaction of viruses with the vesicles under "prefusion" conditions (pH 5; 0 degrees C) and the fusion process itself occurring at elevated temperatures (greater than 15-20 degrees C) only. Despite the observed binding of viruses to LUVs at pH 5 and 0 degrees C, labeling of HA2 was very weak (less than 0.002% of the radioactivity originally present). In contrast, fusion could be readily monitored by the covalent labeling of that polypeptide chain. We have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles. Labeling of the BHA2 polypeptide chain was found to show a remarkable correlation with the temperature dependence of the fusion activity of whole viruses. A temperature-induced structural change appears to be critical for both the interaction of BHA with membranes and the expression of fusion activity of intact viruses.     

Interaction of alpha-crystallin with lens plasma membranes. Affinity for MP26

The binding of the major water-soluble lens protein alpha-crystallin to the lens plasma membrane has been investigated by reassociating purified alpha-crystallin with alpha-crystallin-depleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. alpha-Crystallin reassociates at high affinity (Kd = 13 X 10(-8)M) with alkali-washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles or erythrocyte membranes. Binding to lens plasma membranes is dependent on salt, temperature and pH and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of alpha-crystallin binding suggests the involvement of this transmembrane protein. Binding ist not influenced by pretreatment of membranes with proteases, suggesting that the 4-kDa cytoplasmic fragment of MP26 is not necessary for alpha-crystallin binding. Labeling experiments using (trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine as a probe for intrinsic membrane proteins further showed that alpha-crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized alpha-crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of alpha-crystallin aggregates does not take place in a membrane-bound mode.     

Reconstitution of functional electron transfer between membrane biological elements in a two-dimensional lipidic structure at the electrode interface

These studies develop a methodology to form supported phospholipid bilayers at an electrode/solution interface that models biological membrane systems. Two kinds of electrode were used, a planar gold electrode and a microporous aluminium oxide electrode on which octadecanethiol or octadecyltrichlorosilane was self-assembled. The supported lipidic structures were produced by transfer of a phospholipid monolayer by the Langmuir—Blodgett technique or by direct fusion of phospholipid vesicles. Ubiquinone was introduced into the lipidic structures during their formation; electrochemical measurements demonstrated the mobility of ubiquinone along the plane of the bilayer. A membrane enzyme, pyruvate oxidase from E. coli, was successfully incorporated into this artificial bilayer and was found to be able to exchange electrons with ubiquinone present in the bilayer.     

Parameters affecting fusion between Sendai virus and liposomes. Role of viral proteins, liposome composition, and pH

A kinetic and quantitative characterization of the fusion process between Sendai virus and phospholipid vesicles is presented. Membrane fusion was monitored in a direct and continuous manner by employing an assay which relies on the relief of fluorescence self-quenching of the probe octadecylrhodamine B chloride which was located in the viral membrane. Viral fusion activity was strongly dependent on the vesicle lipid composition and was most efficient with vesicles solely consisting of acidic phospholipids, particularly cardiolipin (CL). This result implies that the fusion of viruses with liposomes does not display an absolute requirement for specific membrane receptors. Incorporation of phosphatidylcholine (PC), rather than phosphatidylethanolamine (PE), into CL bilayers strongly inhibited fusion, suggesting that repulsive hydration forces interfere with the close approach of viral and target membrane. Virus-liposome fusion products were capable of fusing with liposomes, but not with virus. In contrast to fusion with erythrocyte membranes, fusion between virus and acidic phospholipid vesicles was triggered immediately, did not strictly depend on viral protein conformation, and did not display a pH optimum around pH 7.5. On the other hand, with vesicles consisting of PC, PE, cholesterol, and the ganglioside GD1a, the virus resembled more closely the fusogenic properties that were seen with erythrocyte target membranes. Upon decreasing the pH below 5.0, the viral fusion activity increased dramatically. With acidic phospholipid vesicles, maximal activity was observed around pH 4.0, while with GD1a-containing zwitterionic vesicles the fusion activity continued to increase with decreasing pH down to values as low as 3.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence method for measuring the kinetics of fusion between biological membranes

An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biological membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concentrations up to 9 mol% with respect to total lipid, the efficiency of self-quenching is proportional to its surface density. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface density of the fluorophore results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quantitative measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were found to be the same as those determined with a fusion assay based on resonance energy transfer [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099]. Octadecyl Rhodamine B chloride can be readily inserted into native biological membranes by addition of an ethanolic solution of the probe. Evidence is presented showing that the dilution of the fluorophore, occurring when octadecyl Rhodamine containing influenza virus is mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Dilution of the probe was not observed upon prior treatment of fluorescently labeled Sendai virus with trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and interaction with the membrane models of the amino-terminal peptide of influenza virus hemagglutinin HA2 at fusion pH.

Conformations of a 48-mer peptide corresponding to the amino-terminal region of influenza HA2 in aqueous and membranous environments were studied. In aqueous solution the peptide was found to be oligomeric and its helicity was enhanced at higher concentrations. The conformation in phospholipid bilayer and insertion depth into the sodium dodecyl sulfate (SDS) micelle for the fusion peptide were in line with those determined for the amino-terminal 25-mer analog. The turn of residues 28-31 found in the crystal structure of hemagglutinin at neutral pH persisted in the presence of SDS at pH 5.0. Except for the turn, conformational lability of the amino portion of HA2 is suggested by comparison of the secondary structure determined herein with that obtained with the influenza fusion protein crystallized in the aqueous phase at neutral pH. The backbone amide proton exchange experiment suggested an interaction with the micellar surface for the segment carboxy-terminal to the fusion peptide domain.     

Low pH-induced fusion of liposomes with membrane vesicles derived from Bacillus subtilis

We have investigated the pH-dependent interaction between large unilamellar phospholipid vesicles (liposomes) and membrane vesicles derived from Bacillus subtilis, utilizing a fluorescent assay based on resonance energy transfer (RET) (Struck, D. K., Hoekstra, D., and Pagano, R. E. (1981) Biochemistry 20, 4093-4099). Efficient interaction occurs only with negatively charged liposomes, containing cardiolipin or phosphatidylserine, as revealed by the dilution of the RET probes from the liposomal bilayer into the bacterial membrane. The initial rate of fluorophore dilution increases steeply with decreasing pH. The interaction involves a process of membrane fusion, as indicated by the proportional transfer of cholesteryl-[1-14C]oleate, 14C-labeled egg PC, and the RET probes from the liposomes to the bacterial vesicles, the formation of interaction products with an intermediate buoyant density, and the appearance of colloidal gold, initially encapsulated in the liposomes, in the internal volume of fused structures as revealed by thin-section electron microscopy. Treatment of B. subtilis vesicles with trypsin strongly inhibits the fusion reaction, indicating the protein dependence of the process. Vesicles derived from Streptococcus cremoris or from the inner membrane of Escherichia coli also show low pH-dependent fusion with liposomes. The fusion process described in this paper may well be of considerable importance to studies on the mechanisms of membrane fusion and to studies on the structure and function of bacterial membranes. In addition, the fusion reaction could be utilized to deliver foreign substances into bacterial protoplasts.     

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.     

Ca(2+)-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients.

The influence of a transmembrane pH gradient on the Ca(2+)-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets.     

Interaction of liposome with immobilized chitosan during main phase transition

It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on amino-silanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 degrees C and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems.     

Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.     

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.