Cholesterol modulates amiodarone-membrane interactions in model and native membranes

The effects of cholesterol, a lipid mostly found in the sarcolemmal membranes, on the interaction of amiodarone with synthetic models of dimyristoylphosphatidylcholine (DMPC) and with native models of mitochondria and brain microsomes was studied. Alterations on the structural order of lipids were assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probing the bilayer core, and of the propionic acid derivative 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA) probing the outer regions of the bilayer. As detected by the probes and according to classic observations, cholesterol progressively increased the molecular order in the fluid phase of DMPC. Additionally, it modulated the type and extension of amiodarone effects. For low cholesterol concentrations (≤10–15 mol%), amiodarone (50 μM) ordered DMPC bilayers and the effects were almost identical to those observed in pure DMPC. For higher cholesterol concentrations, amiodarone ordering effects decreased slightly and faded for cholesterol concentrations as high as 25 and 30 mol%, when detected by DPH-PA and DPH, respectively. Above these high cholesterol concentrations, a crossover from ordering to disordering effects of amiodarone was apparent, either in the upper region of the bilayer or the hydrophobic core. The effects of amiodarone in native membranes of mitochondria and brain microsomes, in which "native" cholesterol accounts for about 0 and 25 mol%, respectively, correlated reasonably with the results in models of synthetic lipids. There is a close relationship between cholesterol concentration and amiodarone effects, in either synthetic models or native model membranes. Therefore, it may be predicted that the lipid physicochemical properties regulated by cholesterol concentration will also modulate the effects of amiodarone in sarcolemma.     

Non-Selective Toxicological Effects of the Insect Juvenile Hormone Analogue Methoprene. A Membrane Biophysical Approach

The Gram-positive bacterium, Bacillus stearothermophilus, was used as a model organism to identify the non-selective toxic effects of the currently used insecticide methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate). A significant decrease of the yield of bacterial cultures and a premature appearance of ultrastructural abnormalities in cells cultured in the presence of the insecticide were taken as indicators of cytotoxicity. A putative correlation of this cytotoxicity with methoprene-induced perturbations on membrane lipid organization was investigated, using differential scanning calorimetry and the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH-PA). The membrane physical effects depended on the lipid bilayer composition and packing. The most striking effect was a progressive broadening and shifting to lower temperatures, with increasing methoprene concentrations, of the main transition phase of the dimyristoyl- or dipalmitoylphosphatidylcholine bilayers and of the lateral phase separation of liposomes reconstituted with the lipid extracts of B. stearothermophilus.     

Fluorescence study of the fluidity and cooperativity of the phase transitions of zwitterionic and anionic liposomes confined in sol-gel glasses

The current work makes use of different fluorescent reporter molecules and fluorescent spectroscopic techniques to characterize the thermotropic, physical, and dynamical properties of large unilamellar liposomes formed from either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] (DMPG) encapsulated in sol-gel matrixes. In particular, cooperativity of the phase transition is analyzed from steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), the interfacial properties are studied by measuring the spectral shift of Laurdan, and the structural organization (heterogeneity) of the lipid bilayer is determined from the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamical properties in the bulk hydrophobic core is obtained from time-resolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH). The spectroscopic study reveals that upon encapsulation, the basic thermodynamic properties as well as the fluidity of the lipid bilayer practically remain intact for DMPG liposomes but not for DMPC liposomes, whose lipid bilayer exhibits large gel-fluid heterogeneity. On the basis of these experimental results, electrostatic interactions between phospholipid polar heads and the porous surface of the host matrix seem to play a capital role for the preservation of the structural integrity of encapsulated bilayer.     

Chlorin e6-liposome interaction. Investigation by the methods of fluorescence spectroscopy and inductive resonance energy transfer

On the basis of spectral fluorescence and polarization measurements and results obtained on the luminescence quenching of the membrane fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) by incorporated chlorin e6 (chl e6) molecules, it is shown that the interaction of the water-soluble pigment with smaller unilamellar lipid vesicles occurs by a mechanism of partition between the aqueous and lipid phases (partition coefficient Kp = 6.7 x 10(3) and provides rigid fixing of chl e6 monomers at the boundary between the polar and non-polar parts of the lipid membrane. In terms of inductive resonance electronic excitation energy transfer between DPH and chl e6 (R0 = 36.2 A), we have analysed data on DPH fluorescence quenching under different conditions of chl e6 localization in the lipid bilayer and have concluded that the incorporation of the pigment molecules into the vesicles from the aqueous phase occurs mainly into the external monolayer.     

Effect of organotin compounds on membrane lipids: Fluorescence spectroscopy studies

The temperature dependence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) fluorescence anisotropy has been studied to investigate the influence of mono-n-butyltin chloride (MBTC), di-n-butyltin chloride (DBTC) and tri-n-phenyl chloride (TPTC) on the physicochemical state of dipalmitoyl phosphatidylcholine. Below the lipid chain melting transition (T_m), fluorescence anisotropy values of DPH and TMA-DPH are increased by the presence of the organotins, without important modifications of the phase transition temperature. A possible difference in localization of the organotin compounds is suggested by the differential effect of the probes. It is suggested that there is localization in the hydrophobic core of the bilayer for TPTC, and at the head-group level for DBTC, and a homogeneous distribution in the bilayer for MBTC. Similar studies have been performed in liposomal suspensions of cardiolipin, phosphatidylserine and egg phosphatidylcholine.     

Molecular aspects of the interaction between plants sterols and DPPC bilayers: an experimental and theoretical approach

The effect of sterols composition in a lipid bilayer was investigated on membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mixtures with the plant sterols β-sitosterol and stigmasterol. Differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization and infrared spectroscopy studies showed that both sterols changed the packing of the membrane and the hydration of the polar headgroup of the phospholipids, disordering the gel phase and, vice versa, ordering the membrane in the liquid crystalline phase. In all cases some differences among β-sitosterol and stigmasterol could be observed, being β-sitosterol slightly more efficient than stigmasterol in ordering a fluid membrane, bringing the membrane to a more packed liquid ordered phase. Molecular dynamic simulations were carried out to better characterize the distinct behavior of both sterols in a DPPC-membrane. The calculated parameters agreed quite well with the experimental results and a molecular model is proposed to explain differences in the sterols molecules and their effect on the DPPC-bilayer.     

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.     

Liposome fluidization and melting point depression by pressurized CO2 determined by fluorescence anisotropy

The influence of CO2 on the bilayer fluidity of liposomes, which are representative of model cellular membranes, was examined for the first time at the elevated pressures (up to 13.9 MPa) associated with CO2-based processing of liposomes and microbial sterilization. Fluidization and melting point depression of aqueous dipalmitoylphosphatidylcholine (DPPC) liposomes by pressurized CO2 (present as an excess phase) were studied by steady-state fluorescence anisotropy using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Isothermal experiments revealed reversible, pressure-dependent fluidization of DPPC bilayers at temperatures corresponding to near-gel (295 K) and fluid (333 K) phases at atmospheric pressure, where the gel-to-fluid phase transition (Tm) occurs at approximately 315 K. Isobaric measurements (PCO2 =1.8, 7.0, and 13.9 MPa) of DPH anisotropy demonstrate substantial melting point depression (DeltaTm = -4.8 to -18.5 K) and a large broadening of the gel-fluid phase transition region, which were interpreted using conventional theories of melting point depression. Liposome fluidity is influenced by CO2 accumulation in the hydrocarbon core and polar headgroup region, as well as the formation of carbonic acid and/or the presence of buffering species under elevated CO2 pressure.     

Docosahexaenoic acid and eicosapentaenoic acid induce changes in the physical properties of a lipid bilayer model membrane

We investigated the effect of fatty acids such as stearic acid (SA, 18:0), oleic acid (OA, 18:1), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6) on a dipalmitoylphosphatidylcholine (DPPC) bilayer by determining the phase transition temperature, fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), and detergent insolubility. Treatment with unsaturated fatty acid broadened and shifted the phase transitions of the DPPC bilayer to a lower temperature. The phase transition temperature and the value of fluorescence anisotropy of DPH at 37 degrees C decreased progressively with increasing treatment amounts of unsaturated fatty acid. A large amount of the DPPC bilayer treated with unsaturated fatty acid was dissolved in Triton X-100, obtaining a low level of detergent insolubility. These modifications of the bilayer physical properties were most pronounced with DHA and EPA treatment. These data show that unsaturated fatty acids, particularly DHA and EPA, induce a marked change in the lipid bilayer structure. The composition of fatty acids in the DPPC bilayer was similar after treatment with various unsaturated fatty acids, suggesting that the different actions of unsaturated fatty acids are attributed to change in the molecular structure (e.g., kinked conformation by double bonds). We further explored the change in physical properties induced by fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted efficiently on the DPPC bilayer, even when incorporated in emulsion form.     

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.     

Fluorescence studies on radiation oxidative damage to membranes with implications to cellular radiosensitivity

Radiation oxidative damage to plasma membrane and its consequences to cellular radiosensitivity have received increasing attention in the past few years. This review gives a brief account of radiation oxidative damage in model and cellular membranes with particular emphasis on results from our laboratory. Fluorescence and ESR spin probes have been employed to investigate the structural and functional alterations in membranes after y-irradiation. Changes in the lipid bilayer in irradiated unilamellar liposomes prepared from egg yolk lecithin (EYL) were measured by using diphenylhexatriene (DPH) as a probe. The observed increase in DPH polarization and decrease in fluorescence intensity after γ-irradiation of liposomes imply radiation-induced decrease in bilayer fluidity. Inclusion of cholesterol in liposome was found to protect lipids against radiation damage, possibly by modulation of bilayer organization e.g. lipid packing. Measurements on dipalmitoyl phosphatidylcholine (DPPC) liposomes loaded with 6-carboxyfluorescein (CF) showed radiation dose-dependent release of the probe indicating radiation-induced increased permeability. Changes in plasma membrane permeability of thymocytes were monitored by fluorescein diacetate (FDA) and induced intracellular reactive oxygen species (ROS) were determined by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA). Results suggest a correlation between ROS generation and membrane permeability changes induced by radiation within therapeutic doses (0-10 Gy). It is concluded that increase in membrane permeability was the result of ROS-mediated oxidative reactions, which might trigger processes leading to apoptotic cell death after radiation exposure.     

Membrane-forming properties of pseudoglyceryl backbone based gemini lipids possessing oxyethylene spacers

Five pseudoglyceryl backbone based gemini lipids possessing varying lengths of oxyethylene [(-CH2-O-CH2-)n] spacers between cationic ammonium head groups have been synthesized, where n varies from 1 to 5. The membrane-forming properties of these gemini cationic lipids have been investigated. All the gemini lipids formed stable suspensions in water. The presence of membranous aggregates in such lipid suspensions was evidenced by transmission electron microscopy. The membrane-forming characteristics of these gemini lipids were compared with those of the corresponding monomeric lipid with one head group to understand the effect of lipid dimerization. The lipid suspensions were further characterized by dynamic light scattering and zeta potential measurements. Except for the gemini lipid with -CH2-CH2-O-CH2-CH2- spacer (2a), zeta potential of aggregates of all other gemini lipids were significantly greater than that of monomeric lipid suspensions. X-ray diffraction studies with lipid cast films revealed the increase in membrane bilayer width with increase in the length of the spacer (-CH2-O-CH2-)n. Clear thermotropic phase transitions typical of membranous assemblies were observed for all the lipid suspensions by high sensitivity differential scanning calorimetry. Aggregates of gemini lipid 2a bearing one oxyethylene [-(CH2-CH2-O-CH2-CH2)-] unit between headgroups manifested the highest phase transition temperature as compared to other gemini analogues as well as that of monomeric lipid 1. The phase transitions were reversible and exhibited large hysteresis, indicating that the observed phase transitions were of first order. To probe the surface hydration of these membranous aggregates, Paldan fluorescence studies were performed. These studies indicated the high polarity of the vesicular surface of gemini lipid 2a both in the gel and fluid melted phase as compared to vesicles of other gemini lipids.     

Relationship between lipid peroxidation and rigidity in L-alpha-phosphatidylcholine-DPPC vesicles

DPPC incorporation into egg-PC unilamellar vesicles reduces their oxidation rate beyond that expected from the unsaturated lipid dilution. Addition of the unsaturated lipids produces changes in the physical properties of the inner parts of the lipid bilayer, as sensed by fluorescence anisotropy of DPH, and in the hydrophilic/hydrophobic region, as sensed by the generalized polarization of laurdan. DPPC (30 mol%) incorporation into egg-PC vesicles produces a decrease in alkyl chain mobility in the inner part of the bilayer, evaluated by the increase of DPH fluorescence anisotropy, and a rise of the generalized polarization value of laurdan in the bilayer interface. It also leads to a decrease in the rate of water efflux promoted by a hypertonic shock. Oxidation of PC LUVs, promoted by AAPH, as sensed by oxygen uptake and MDA formation, leads to qualitatively similar results than DPPC addition: rigidification at the inner part and the surface of the liposomes, and a lower rate of water permeation. It is suggested that these changes could contribute to the observed decrease in oxidation rate with conversion.     

Immobilizing single lipid and channel molecules in artificial lipid bilayers with annexin A5

The effects of annexin A5 on the lateral diffusion of single-molecule lipids and single-molecule proteins were studied in an artificial lipid bilayer membrane. Annexin A5 is a member of the annexin superfamily, which binds preferentially to anionic phospholipids in a Ca2+-dependent manner. In this report, we were able to directly monitor single BODIPY 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules in lipid bilayers containing phosphatidylserine (PS) by using fluorescence microscopy. The diffusion coefficients were calculated at various annexin A5 concentrations. The diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 in the absence of annexin A5 were 4.81 x 10(-8) cm(2)/s and 2.13 x 10(-8) cm(2)/s, respectively. In the presence of 1 microM annexin A5, the diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 were 2.2 x 10(-10) cm(2)/s and 9.5 x 10(-11) cm(2)/s, respectively. Overall, 1 microM of annexin A5 was sufficient to induce a 200-fold decrease in the lateral diffusion coefficient. Additionally, we performed electrophysiological examinations and determined that annexin A5 has little effect on the function of RyR2. This means that annexin A5 can be used to immobilize RyR2 in a lipid bilayer when imaging and analyzing RyR2.     

Cardiolipin, a key component to mimic the E. coli bacterial membrane in model systems revealed by dynamic light scattering and steady-state fluorescence anisotropy

The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA-DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for Escherichia coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared with those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.     

Immobilised artificial membrane chromatography coupled with molecular probing. Mimetic system for studying lipid-calcium interactions in nutritional mixtures

Immobilised artificial membrane (IAM) chromatography was utilised to study the interactions of usual membrane probes with grafted phosphatidylcholine silica support, in relation to the presence of calcium ions introduced in the mobile phase as they are present in nutritional mixtures. IAM acts as a mimetic membrane of lipid emulsion globules, a major component of nutritional mixtures. The tested probes were 1,6-diphenyl-1,3,5-hexatriene (DPH), 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one or nile red (NR) and 2-(p-toluidinyl)naphtalene-6-sulfonate (TNS). For each probe, partition coefficients and thermodynamic parameters of transfer from the mobile phase to the IAM stationary phase have been measured. Our results suggested that the interactions of neutral probes (i.e. DPH and NR) with phosphatidylcholine are driven by hydrophobic forces. Addition of calcium chloride to the mobile phase slightly decreased the retention of these neutral probes and dramatically increased that of anionic TNS. Moreover, an enthalpy-entropy compensation study revealed that the mechanism of interaction between TNS and IAM is independent of the calcium concentration. Results argued for the existence of electrostatic repulsion forces exerted by IAM phase towards anionic TNS. Addition of calcium ions into the mobile phase led to the establishment of an ionic double layer at the zwitterionic stationary phase surface weakening the electrostatic barrier and increasing TNS retention. Consequently, it was demonstrated that IAM appears as a suitable model to get a better insight on the lipid-calcium interactions taking place in nutritional mixtures.     

Interfacial interactions between poly[L-lysine]-based branched polypeptides and phospholipid model membranes

The interaction of five poly[L-lysine]-derived branched chain polypeptides of poly[Lys(X(i))] (X(i)K) or poly[Lys(X(i)-DL-Ala(m))] (XAK) with lipid bilayers (DPPC and DPPC/PG, 8:2) was studied by fluorescence polarization techniques. Two fluorescent probes, DPH and TMA-DPH, were utilized to monitor changes of motion in the internal and/or in the polar head regions, respectively. Results indicate that the interaction of polypeptides with neutral (DPPC) bilayers is mainly dependent on the polarity and electrical charge of side chains. The amphoteric E(i)K shows the highest level of interaction. Polycationic polypeptides (H(i)K, P(i)K, TAK) have a relatively small effect on the transition temperature of the lipids, while the polyanionic Succ-EAK has no effect at the alkyl chain region of the bilayer. Data with TMA-DPH indicate the lack of pronounced interaction between the polypeptides and the outer surface of the liposome. Similar tendency was documented for DPPC/PG vesicles. Polypeptides, H(i)K, and P(i)K induce significant changes in the transition temperature, thus indicating their insertion into the hydrophobic core of the bilayer without marked effect on the polar head region. Results suggest that these polypeptides (except E(i)K) have no destabilizing effect on liposomes studied. These properties are considered as beneficial for their use as safe carriers for bioactive molecules.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Aqueous dispersions of cubic lipid–water phases

Inverse lipid–water phases such as cubic phases can form kinetically stable dispersions by fragmentation in water. Cubic lipid phases can be dispersed by polar lipids favoring lamellar phases or by block copolymers, which can close the bilayer at the surface so that the hydrocarbon chain core is not exposed to water. Monodisperse particles based on glycerol monooleate, with their bilayer curved as the P-, D- or G-minimal surface, have been prepared in this way. Their inner bilayer conformation and outer shape have been examined, mainly by X-ray diffraction and cryo transmission electron microscopy. There is also a different type of cubic lipid bilayer particles with a periodicity in the micrometer range, which have been identified in phospholipid–water dispersions and in cell membrane assemblies. The mechanism behind formation in vivo of such cubic membranes, which also follow the P-, D- and G-surfaces, is discussed. Other lipid–water dispersions with lower symmetry are finally considered; dispersions formed by the inverse hexagonal phase and the dispersed state of a tetragonal bilayer structure formed by lung surfactants.     

Spontaneously formed vesicles of sodium N-(11-acrylamidoundecanoyl)-glycinate and L-alaninate in water

Two N-acyl amino acid surfactants, sodium N-(11-acrylamidoundecanoyl)-glycinate (SAUG) and L-alaninate (SAUA), were synthesized and characterized in aqueous solution. A number of techniques, such as surface tension, fluorescence probe, light scattering, and transmission electron microscopy were employed for characterization of the amphiphiles in water. The surface and interfacial properties were measured. The amphiphiles have two critical aggregation concentrations. The results of surface tension and fluorescence probe studies suggested formation of bilayer self-assemblies in dilute aqueous solutions of the amphiphiles. The magnitudes of free energy change of aggregation have indicated that bilayer formation is more favorable in the case of SAUG. Steady-state fluorescence measurements of pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to study the microenvironment of the molecular self-assemblies. Temperature-dependent fluorescence anisotropy change of DPH probe revealed phase transition temperature of the bilayer self-assemblies. The effects of pH on the structure of the self-assemblies of SAUG and SAUA have been studied. The role of intermolecular hydrogen bonding between amide groups upon aggregation toward microstructure formation in solution has been discussed. Circular dichroism spectra suggested the presence of chiral aggregates in an aqueous solution of SAUA. The transmission electron micrographs revealed the presence of closed spherical vesicles in aqueous solutions of the amphiphiles. Dynamic light scattering measurements were performed to obtain average size of the aggregates.