Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

The influence of the molar ratio of cholesteryl hemisuccinate/dipalmitoylphosphatidylcholine on 'liposome' formation after lipid film hydration.

The morphology of the structures formed after hydration of lipid films of cholesteryl hemisuccinate/dipalmitoylphosphatidylcholine (CHEMS/DPPC) was investigated in low ionic strength solutions. The importance of addition of a charge inducing agent/geometrical structure such as CHEMS for the formation of stable vesicle dispersions upon hydration was demonstrated. The encapsulated volume measured for CHEMS/DPPC ratios below 1:50 was low. For a ratio of CHEMS/DPPC of 1:30 EM micrographs showed mainly small unilamellar vesicles, with particle sizes between 0.07 and 0.3 microns, together with a small number of much larger vesicles. For ratios of CHEMS/DPPC above 0.1 only unilamellar vesicles and no bilayer stacks were found. The results confirm the hypothesis by Hauser (Biochim. Biophys. Acta 772 (1984) 37-50), that the structures formed upon hydration of charged phospholipid films are unilamellar vesicles, while for neutral phospholipid films upon hydration bilayer stacks and multilamellar vesicles are formed. The effect of CHEMS on the liposome bilayer structure can be mainly ascribed to its charge inducing properties and presumably to a minor extent to its molecular geometry, or to a combination of both.     

Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.     

Phase behaviour of a diglyceride prodrug: spontaneous formation of unilamellar vesicles

A prodrug (Fig. 1(IV)) is synthesized consisting of the beta-blocker bupranolol which is covalently linked to 1, 3-dipalmitoyl-2-succinyl-glycerol. The resulting lipid-like prodrug is amphipathic and surface active. It disperses readily in H2O above 30 degrees C forming a smectic lamellar phase. This prodrug bears one positive charge at neutral pH and hence the swelling behaviour of dispersions in H2O is similar to that of charged phospholipids: the dispersions show continuous swelling with increasing water content and consequently in the excess H2O region of the phase diagram the thermodynamically most stable structure is the unilamellar vesicle. This includes oligomeric vesicles which may be defined as unilamellar vesicles containing smaller, also unilamellar vesicles entrapped in their internal aqueous compartment. The prodrug dispersions in H2O are polydisperse with vesicle sizes ranging from 0.1 micron to several micron. Sonication of these dispersions produce small unilamellar vesicles of an average size and size distribution similar to sonicated egg phosphatidylcholine dispersions. Unsonicated dispersions of the prodrug in H2O undergo reversibly sharp order-disorder transitions at 32 degrees C with an enthalpy change of delta H = 10 kcal/mol. In sonicated aqueous dispersions this phase transition is asymmetric and significantly broadened indicating that the cooperativity is markedly reduced. The peak temperature and enthalpy change of this broad transition are reduced compared to the transition observed with unsonicated dispersions. The temperature dependence of the electron spin resonance (ESR) hyperfine splitting and order parameter also reflects the order-disorder transition. From ESR spin labeling it is concluded that in sonicated dispersions the prodrug molecule is more mobile and its anisotropy of motion is reduced compared to unsonicated dispersions. This result indicates that the molecular packing in the highly curved bilayers of small unilamellar prodrug vesicles is significantly perturbed compared to bilayers of unsonicated dispersions.     

Early steps of supported bilayer formation probed by single vesicle fluorescence assays

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Methylenetetrahydrofolate-dependent biosynthesis of ribothymidine in transfer RNA of Streptococcus faecalis. Evidence for reduction of the 1-carbon unit by FADH2

The methyl carbon of ribothymidine in Loop IV of the tRNA of Streptococcus faecalis, Bacillus subtilis, and some other microorganisms is derived directly from 5,10-methylenetetrahydrofolate, not S-adenosylmethionine. The pure enzyme from S. faecalis also requires FADH2. We have obtained evidence that tetrahydrofolate is a product of the reaction and demonstrated that label from [5-3H]5-deazaFMNH2 is incorporated into the methyl moiety of ribothymidine. These data indicate that the enzyme uses methylenetetrahydrofolate solely as a 1-carbon donor and employs FADH2 as a reducing agent in vitro according to the following reaction: tRNA(U psi C) + CH2 = THF + FADH2 leads to tRNA(T psi C) + THF + FAD.     

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of electrolyte on the morphology of vesicles composed of the dialkyl polyoxyethylene ether surfactant 2C18E12

Small-angle neutron-scattering (SANS) studies were performed on vesicles composed of 1,2-di-O-octadecyl-rac-glyceryl-3-(omega-methoxydodecaethylene glycol), in deuterium oxide (D2O) solutions with various ionic strengths of LiCl, NaCl and NaI. Gross vesicle morphologies, examined using freeze-fracture electron microscopy, showed that NaCl promoted the formation of multilamellar vesicles. Model fitting of the SANS data showed changes in bilayer parameters such as thickness and repeat spacings, in response to the presence of ions in the bulk solution. 2C18E12 vesicles in D2O are shown to exist as predominantly unilamellar structures with a bilayer thickness of approximately 51 A. Vesicles in increasing concentrations of LiCl and NaCl exhibit decreased layer thickness and increased lamelarity. Little change was observed for vesicles formed in NaI solutions. We suggest that these changes result from intrusion of E12 headgroups into the alkyl chain region of the vesicle bilayers, in response to the increase in concentration of ions present and their charge density.     

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.     

Ca2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles

The effect of cholesterol on the Ca2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c11, and of the fusion reaction itself, f11. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c11 and f11 increase steeply with the Ca2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c11 decreases, while the rate of the actual fusion reaction, f11, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.     

Effect of cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide.

The effect of cholesterol on the bilayer partitioning of the peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) and its assembly into a pore in large unilamellar vesicles composed of neutral and negatively charged phospholipids has been determined. GALA undergoes a conformational change from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0, inducing at low pH leakage of contents from vesicles. Leakage from neutral or negatively charged vesicles at pH 5.0 was similar and could be adequately explained by the mathematical model (Parente, R. A., S. Nir, and F. C. Szoka, Jr., 1990. Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. Biochemistry. 29:8720-8728) which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of 10 +/- 2 peptides. Increasing cholesterol content in the membranes resulted in a reduced efficiency of the peptide to induce leakage. Part of the cholesterol effect was due to reduced binding of the peptide to cholesterol-containing membranes. An additional effect of cholesterol was to increase reversibility of surface aggregation of the peptide in the membrane. Results could be explained and predicted with a model that retains the same pore size, i.e., 10 +/- 2 peptides, but includes reversible aggregation of the monomers to form the pore. Resonance energy transfer experiments using fluorescently labeled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol-containing liposomes, thus reducing the efficiency of pore formation.     

Reaction characteristics and mechanisms of lipid bilayer vesicle fusion

Small unilamellar lipid bilayer vesicles were prepared from brain phosphatidylserine, egg phosphatidylcholine, and synthetic dipalmitoylphosphatidylcholine, and were fused into larger structures by freezing and thawing, addition of calcium chloride, and passage through the lipid phase transition temperature. Fusion reactions were studied by electron microscopy, light scattering, and use of fluorescent probes. Fusion was accompanied by leakage of lipid vesicle constituents and of water-soluble solutes in the inner vesicle compartments, and by uptake of these types of components from the external solution. Such leakage was greater during fusion by freezing than by Ca²⁺. Passage through the transition temperature produced a moderate degree of fusion, without loss of membrane components. It is concluded that each fusion method gives rise to a characteristic size or narrow range of sizes of fusion products. The fraction of small vesicles fused into larger structure depends on the method of vesicle preparation, composition of the lipid bilayer, and composition of the external solution. Fusion is induced by creation of a discontinuity in the bilayer or by removal of water associated with the bilayer. The amount of water removed controls the extent of fusion. This is maximized in bilayers when in the liquid-crystal phase, as against the gel phase, in vesicles made by ethanol injection, as against sonication, and in charged bilayers, as against neutral ones.     

Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction

Cholesterol sulphate is a potent stabilizer of membrane bilayer structure in both dielaidoylphosphatidylethanolamine and egg phosphatidylethanolamine model membranes, however, the addition of calcium abolishes this bilayer stabilization. Calcium also induces fusion and leakage of egg phosphatidylethanolamine large unilamellar vesicles containing cholesterol sulphate, but has no effect on fusion or leakage of egg phosphatidylcholine large unilamellar vesicles containing cholesterol sulphate. With egg phosphatidylethanoiamine liposomes, the initial rate, and extent of fusion, at constant calcium concentration, vary inversely with the mol percentage of cholesterol sulphate present in the vesicle membrane. The interaction of calcium and cholesterol sulphate, which causes membrane destabilization and fusion in phosphatidylethanolamine containing model systems, may play a role in the acrosome reaction in human sperm.     

Gramicidin induced aggregation and size increase of phosphatidylcholine vesicles

To investigate the role of membrane proteins in the fusion process, linear hydrophobic polypeptide gramicidin was used as fusogenic agent in small unilamellar vesicles (SUV) constituted of saturated lecithins. It was found that gramicidin, externally added to a suspension of vesicles, induces a reversible vesicles aggregation. When incorporated into the bilayer, gramicidin induces increase in vesicle size. The vesicle size increase was monitored by column chromatography and transmission electron microscopy. The process of vesicle size increase occurs only when the lipid membrane is in the gel state. A maximum is observed in the kinetics at a temperature of approx. 25 degrees C lower than the phase transition temperature of lipids. Higher rates of vesicle size increase are obtained as the lipid chain length increases. The process is accompanied by a release of internal vesicle content and by membrane lipid mixing.     

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.     

Surface specific kinetics of lipid vesicle adsorption measured with a quartz crystal microbalance.

We have measured the kinetics of adsorption of small (12.5-nm radius) unilamellar vesicles onto SiO2, oxidized gold, and a self-assembled monolayer of methyl-terminated thiols, using a quartz crystal microbalance (QCM). Simultaneous measurements of the shift in resonant frequency and the change in energy dissipation as a function of time provide a simple way of characterizing the adsorption process. The measured parameters correspond, respectively, to adsorbed mass and to the mechanical properties of the adsorbed layer as it is formed. The adsorption kinetics are surface specific; different surfaces cause monolayer, bilayer, and intact vesicle adsorption. The formation of a lipid bilayer on SiO2 is a two-phase process in which adsorption of a layer of intact vesicles precedes the formation of the bilayer. This is, to our knowledge, the first direct evidence of intact vesicles as a precursor to bilayer formation on a planar substrate. On an oxidized gold surface, the vesicles adsorb intact. The intact adsorption of such small vesicles has not previously been demonstrated. Based on these results, we discuss the capacity of QCM measurements to provide information about the kinetics of formation and the properties of adsorbed layers.     

A catalytic intermediate and several flavin redox states stabilized by folate-dependent tRNA methyltransferase from Bacillus subtilis

The flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH(2)THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH(?) radical, and a catalytic intermediate endowed with methylating activity. The FADH(?) radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH(2)THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH(2)THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH(2)THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions.