The effects of 2H2O on the phase transition of large unilamellar vesicle (LUV) suspensions as detected by ultrasound spectroscopy and electron spin resonance

The importance of water in the molecular dynamics of large unilamellar vesicle (LUV) suspensions, in which increasing portions of the water were replaced by 2H2O, was investigated. Determinations of the ultrasonic absorption coefficient per wavelength, alpha lambda, were performed as a function of temperature and frequency for LUVs (LUVs: 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine, DPPC, and dipalmitoylphosphatidylglycerol, DPPG) in the vicinity of their phospholipid phase transition, using a double crystal acoustic interferometer. Electron spin resonance (ESR) and differential scanning calorimetry (DSC) were also employed to probe this system. When increasing portions of the aqueous content of the LUV suspensions were replaced by 2H2O the phase transition temperature increased from 42.0 degrees C to 42.9 degrees C (indicating an increase in the activation energy of the transition), and the amplitude of alpha lambda at the phase transition increased. However, alpha lambda max as a function of frequency at the phase transition did not change with the addition of 2H2O, indicating that the relaxation time of the event responsible for the absorption of ultrasound was unaffected. The increase in the activation energy of the transition with the addition of 2H2O suggested that the mobility of phospholipids near the membrane/aqueous interface was changed. Electron spin resonance (ESR) experiments on LUVs with nitroxide spin probes positioned at the membrane/aqueous interface (5-doxyl stearate and CAT16) showed that LUVs in 2H2O have a broader splitting, Amax, at the membrane/aqueous interface than do LUVs in H2O. These results suggest that 2H2O changes the mobility and/or structure of the phospholipids in the region of the membrane/aqueous interface. This difference in Amax was not seen for the probe PC-12-doxyl stearate, which resides at the C-12 position of the bilayer.     

Ultrasound interaction with large unilamellar vesicles at the phospholipid phase transition: perturbation by phospholipid side chain substitution with deuterium

The ultrasonic absorption, alpha lambda, as a function of temperature and frequency was determined in large unilamellar vesicles (LUVs) in which specific phospholipid side chains were deuterated. Deuteration significantly altered the temperature and frequency dependence of alpha lambda. The frequency change was especially marked, with decreased frequency and broadening of the ultrasound relaxation, even with only minor changes in the phase transition temperature. Deuteration decreased the Tm and enthalpy of the lipid phase transition, as shown by differential scanning calorimetry, whereas electron spin resonance showed that at and above the lipid phase transition, no differences in the mobility as a function of temperature were observed. These results show that the observed increase in ultrasonic absorption in LUVs at the phospholipid phase transition arises from the interaction of ultrasound with the hydrophobic side chains, probably coupling with structural reorganization of small domains of molecules, a process which is maximized at the phase transition temperature.     

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.     

Study of the interaction of an alpha-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic alpha-helical transmembrane peptide, acetyl-K(2)-L(24)-K(2)-amide (L(24)), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L(24) decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L(24) in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L(24):PC=1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L(24). As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and (2)H NMR spectroscopy study of the interaction of the L(24) and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between (2)H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L(24) suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.     

Ultrasonic evidence for structural relaxation in large unilamellar liposomes

The ultrasonic absorption of large unilamellar vesicles (average diameter 0.2 micron) was determined in the frequency range 0.5-5 MHz. The liposomes were composed of a 4:1 mixture by weight of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol. They were studied with and without cholesterol or gramicidin incorporated into the bilayer. A large increase in absorption occurs at the solid to liquid-crystalline phase transition temperature (42 degrees C) of the pure lipid vesicles. This increase in absorption is interpreted as a structural relaxation of the 'melting' fatty acid chains occurring with an average relaxation time of 76 ns. The liposomes were also found to be extremely permeable near the transition temperature. Essentially complete release of cytosine arabinoside, a small water-soluble molecule, occurred at 42 degrees C. Addition of cholesterol or gramicidin to the bilayer of the liposomes broadened the ultrasonic absorption and reduced the efflux of cytosine arabinoside at the phase transition. No increase in absorption was observed at the transition temperature in the presence of 50 mol% of cholesterol. Gramicidin, in addition to broadening the transition, slows the isomerization of bonds in the hydrocarbon chains of the lipids. A concentration of 5 mol% gramicidin increased the average relaxation time to 211 ns.     

Solute effects on the colloidal and phase behavior of lipid bilayer membranes: ethanol-dipalmitoylphosphatidylcholine mixtures.

By means of the scanning differential calorimetry, x-ray diffractometry, and the dynamic light scattering, we have systematically studied the phase and packing properties of dipalmitoylphosphatidylcholine vesicles or multibilayers in the presence of ethanol. We have also determined the partial ternary phase diagram of such dipalmitoylphosphatidylcholine/water/ethanol mixtures. The directly measured variability of the structural bilayer parameters implies that ethanol binding to the phospholipid bilayers increases the lateral as well as the transverse repulsion between the lipid molecules. This enlarges the hydrocarbon tilt (by up to 23 degrees) and molecular area (by < or = 40%). Ethanol-phospholid association also broadens the interface and, thus, promotes lipid headgroup solvation. This results in excessive swelling (by 130%) of the phosphatidylcholine bilayers in aqueous ethanol solutions. Lateral bilayer expansion, moreover, provokes a successive interdigitation of the hydrocarbon chains in the systems with bulk ethanol concentrations of 0.4-1.2 M. The hydrocarbon packing density as well as the propensity for the formation of lamellar gel phases simultaneously increase. The pretransition temperature of phosphatidylcholine bilayers is more sensitive to the addition of alcohol (initial shift: delta Tp = 22 degrees C/mol) than the subtransition temperature (delta Ts reversible 5 degrees C/mol), whereas the chain-melting phase transition temperature is even less affected (delta Tm = 1.8 degrees C/mol). After an initial decrease of 3 degrees for the bulk ethanol concentrations below 1.2 M, the Tm value increases by 2.5 degrees above this limiting concentration. The gel-phase phosphatidylcholine membranes below Tm are fully interdigitated above this limiting concentration. The chain tilt on the fringe of full chain interdigitation is zero and increases with higher ethanol concentrations. Above Tm, some of the lipid molecules are solubilized by the bound ethanol molecules. More highly concentrated ethanol solutions (> 7 M) solubilize the phosphatidylcholine bilayers with fluid chains fully and result in the formation of mixed lipid-alcohol micelles.     

Very high frequency electron paramagnetic resonance of 2,2,6,6-tetramethyl-1-piperidinyloxy in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine liposomes: partitioning and molecular dynamics.

Partitioning and molecular dynamics of 2,2,6,6,-tetramethylpiperedine-1-oxyl (TEMPO) nitroxide radicals in large unilamellar liposomes (LUV) composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine were investigated by using very high frequency electron paramagnetic resonance (EPR) spectroscopy. Experiments carried out at a microwave frequency of 94.3 GHz completely resolved the TEMPO EPR spectrum in the aqueous and hydrocarbon phases. An accurate computer simulation method combined with Levenberg-Marquardt optimization was used to analyze the TEMPO EPR spectra in both phases. Spectral parameters extracted from the simulations gave the actual partitioning of the TEMPO probe between the LUV hydrocarbon and aqueous phases and allowed analysis of picosecond rotational dynamics of the probe in the LUV hydrocarbon phase. In very high frequency EPR experiments, phase transitions in the LUV-TEMPO system were observed as sharp changes in both partitioning and rotational correlation times of the TEMPO probe. The phase transition temperatures (40.5 +/- 0.2 and 32.7 +/- 0.5 degrees C) are in agreement with previously reported differential scanning microcalorimetry data. Spectral line widths were analyzed by using existing theoretical expressions for motionally narrowed nitroxide spectra. It was found that the motion of the small, nearly spherical, TEMPO probe can be well described by anisotropic Brownian diffusion in isotropic media and is not restricted by the much larger hydrocarbon chains existing in ripple structure (P beta') or fluid bilayer structure (L alpha) phases.     

A comparative study of the phase transitions of phospholipid bilayers and monolayers

Phase transitions in bilayers and monolayers of various synthetic phospholipids with different chain lengths as well as different polar head groups were studied by differential scanning calorimetry or with the film balance technique, respectively. With the film balance, area versus temperature curves (isobars) were recorded at different surface pressures. The monolayer phase transition from the fluid-condensed to the fluid-expanded phase is shifted towards higher temperature when the lateral pressure in the monolayer is increased. The temperature dependence of the equilibrium pressure as well as the magnitude of the area change at the transition depends only on the nature of the phospholipid head group and not on the chain length of the hydrocarbon chains of the lipid. Phospholipids with strong intermolecular attractive interactions between the head groups show low values for dpi/dTm and for the area change, deltaf, whereas phospholipids with negatively charged head groups without intermolecular attractive forces exhibit higher values for dpi/dTm and deltaf. The shift of the monolayer phase transition temperature when increasing the chain length of the lipid is almost identical to the shift in Tm observed for the bilayer system of the same phospholipids. A comparison of monolayer and bilayer systems on the basis of the absolute value of the molecular area of the phospholipid in the bilayer gel phase and the change in area at the bilayer and monolayer transition leads to the following conclusions. The behaviour of the bilayer system is very similar to that of the respective monolayer system at a lateral pressure of approx. 30 dyne/cm, because at this pressure the absolute area and the area change in both systems are the same. Further support for this conclusion comes from the experimental finding that a lateral pressure of 30 dyne/cm the shift in Tm due to the increase in charge when the methyl ester of phosphatidic acid is investigated is the same for the bilayer and the monolayer system.     

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and ²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between ²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Calorimetric, x-ray diffraction, and spectroscopic studies of the thermotropic phase behavior and organization of tetramyristoyl cardiolipin membranes

The thermotropic phase behavior and organization of aqueous dispersions of the quadruple-chained, anionic phospholipid tetramyristoyl diphosphatidylglycerol or tetramyristoyl cardiolipin (TMCL) was studied by differential scanning calorimetry, x-ray diffraction, (31)P NMR, and Fourier-transform infrared (FTIR) spectroscopy. At physiological pH and ionic strength, our calorimetric studies indicate that fully equilibrated aqueous dispersions of TMCL exhibit two thermotropic phase transitions upon heating. The lower temperature transition is much less cooperative but of relatively high enthalpy and exhibits marked cooling hysteresis, whereas the higher temperature transition is much more cooperative and also exhibits a relatively high enthalpy but with no appreciable cooling hysteresis. Also, the properties of these two-phase transitions are sensitive to the ionic strength of the dispersing buffer. Our spectroscopic and x-ray diffraction data indicate that the lower temperature transition corresponds to a lamellar subgel (L(c)') to gel (L(beta)) phase transition and the higher temperature endotherm to a L(beta) to lamellar liquid-crystalline (L(alpha)) phase transition. At the L(c)'/L(beta) phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from approximately 11 A to approximately 50 A, and this value decreases slightly at the L(beta)/L(alpha) phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8 A to 43.5 A at the L(c)'/L(beta) phase transition, consistent with the loss of the hydrocarbon chain tilt of approximately 12 degrees , and decreases to 37.8 A at the L(beta)/L(alpha) phase transition. The calculated cross-sectional areas of the TMCL molecules are approximately 79 A(2) and approximately 83 A(2) in the L(c)' and L(beta) phases, respectively, and we estimate a value of approximately 100 A(2) in the L(alpha) phase. The combination of x-ray and FTIR spectroscopic data indicate that in the L(c)' phase, TMCL molecules possess tilted all-trans hydrocarbon chains packed into an orthorhombic subcell in which the zig-zag planes of the chains are parallel, while in the L(beta) phase the untilted, all-trans hydrocarbon chains possess rotational mobility and are packed into a hexagonal subcell, as are the conformationally disordered hydrocarbon chains in the L(alpha) phase. Our FTIR spectroscopic results demonstrate that the four carbonyl groups of the TMCL molecule become progressively more hydrated as one proceeds from the L(c)' to the L(beta) and then to the L(alpha) phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our (31)P-NMR results indicate that although the polar headgroup retains some mobility in the L(c)' phase, its motion is much more restricted in the L(beta) and especially in the L(alpha) phase than that of other phospholipids. We can explain most of our experimental results on the basis of the relatively small size of the polar headgroup of TMCL relative to other phospholipids and the covalent attachment of the two phosphate moieties to a single glycerol moiety, which results in a partially immobilized polar headgroup that is more exposed to the solvent than in other glycerophospholipids. Finally, we discuss the biological relevance of the unique properties of TMCL to the structure and function of cardiolipin-containing biological membranes.     

Interaction of synthetic glycophospholipids with phospholipid bilayer membranes.

A series of glycophospholipids synthesized by coupling mono-, di-, or tri-saccharides to dioleoylphosphatidylethanolamine (DOPE) by reductive amination was used to investigate the interaction of glycophospholipids with phospholipid bilayer membranes. These synthetic glycophospholipids functioned as a stabilizer for the formation of DOPE bilayer vesicles. The minimal mol% of glycophospholipid needed to stabilize the DOPE vesicles were as follows: 8% N-neuraminlactosyl-DOPE (NANL-DOPE), 20% N-maltotriosyl-DOPE (MAT-DOPE), 30% N-lactosyl-DOPE (Lac-DOPE), and 42% N-galactosyl-DOPE (Gal-DOPE). The estimated hydration number of glycophospholipid in reverse micelles was 87, 73, 46, and 14 for NANL-DOPE, MAT-DOPE, Lac-DOPE, and Gal-DOPE, respectively. Thus, the hydration intensity of the glycophospholipid was directly related to the ability to stabilize the DOPE bilayer phase for vesicle formation. Glycophospholipids also reduced the transition temperature from gel to liquid-crystalline phase (Tm) of dipalmitoylphosphatidylcholine (DPPC) bilayers. Interestingly, incorporation of NANL-DOPE induced a decrease of membrane fluidity of DPPC bilayers in the gel phase while other glycophospholipids had no effect. Also, low level of NANL-DOPE but not other glycophospholipids increased the transition temperature (TH) from liquid-crystalline to hexagonal phase of dielaidoylphosphatidylethanolamine bilayers. These results showed that NANL-DOPE with a highly hydratable headgroup which provides a strong stabilization activity for the L alpha phase of phospholipid membranes, may also be involved in specific interactions with neighboring phospholipids via its saccharide moiety.     

Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters.

Thermozeaxanthins (TZS) are novel carotenoid-glucoside esters existing in the cell membranes of the thermophilic bacterium, Thermus thermophilus. The effect of TZS on membrane permeability was studied by measuring the leakage of the fluorescent dye from calcein-entrapped large unilamellar liposomes (LUVs). The LUVs were composed of a small portion (0.2-1.0 mol%) of TZS and phosphatidylcholine (PC) of various length and saturation degree of hydrocarbon chains. Incorporation of TZS in egg PC LUVs stabilized the liposomes in the temperature range from 30 to 80 degrees C, as only 2.6% of the entrapped calcein leaked out in contrast to 10% release from the egg PC liposomes without TZS. LUVs composed of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) were stabilized by the incorporation of TZS at a temperature below 30 degrees C. Inclusion of TZS in LUVs composed of dimyristoylphosphatidylcholine, whose hydrocarbon chains are shorter than both DPPC and DOPC, did not stabilize the liposomes. About 90% of the entrapped dye was lost indicating defects of the liposomal membranes. Matching of the lipid bilayer thickness with the molecular length of TZS in the bilayers is discussed.     

Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, laurdan generalized polarization and carboxyfluorescein leakage studies

The perturbation of the lipid bilayer structure by tamoxifen may contribute to its multiple mechanisms of anticancer action not related to estrogen receptors. This study evaluates the effect of tamoxifen on structural characteristics of model membranes using differential scanning calorimetry (DSC), fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-[trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), as well as 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) generalized polarization. The comparative measurements in multilammelar vesicles (MLV) prepared from dipalmitoylphosphatidylcholine (DPPC) revealed that tamoxifen decreases the phase transition temperature (Tm) paralleled by a broadening of the phase transition profile. In large unilamellar vesicles (LUV) prepared from egg yolk phosphatidylcholine (EPC), tamoxifen increased the lipid bilayer order predominantly in the outer bilayer region. From membrane permeability measurements, we conclude that the tamoxifen-induced release of entrapped carboxyfluorescein (CF) results from a permanent bilayer disruption and the formation of transient holes in the lipid bilayer.     

Thermotropic and dynamic characterization of interactions of acylated alpha-bungarotoxin with phospholipid bilayer membranes

The interactions of palmitoyl-alpha-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (Tm), a broadening of the heat capacity function, and a decrease in the enthalpy change associated with the phospholipid gel to liquid-crystalline transition. Analysis of the dependence of this decreased enthalpy change on the protein/lipid molar ratio indicates that each PBGT molecule exhibits a localized effect upon the bilayer, preventing approximately six lipid molecules from participating in the lipid phase transition. Additional calorimetric experiments indicate that binding to acetylcholine receptor enriched membranes causes a small increase in the Tm of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below Tm while increasing the order above Tm. These results have been further supported by rotational mobility measurements of erythrosin-labeled PBGT associated with giant (about 2-micron) unilamellar vesicles composed of dielaidoylphosphatidylcholine or dioleoylphosphatidylcholine using the time-dependent decay of delayed fluorescence/phosphorescence emission anisotropy. Rotational correlation times in the submillisecond time scale (about 30 microseconds) indicate that the protein is highly mobile in the fluid phase and that below Tm the rotational mobility is only slightly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Effects of phenylamide herbicides on the physical properties of phosphatidylcholine membranes

A number of phenylamide herbicides are observed to uncouple electron transport in isolated chloroplasts and mitochondria and alter the H+ permeability of artificial liposomes. Several of these phenylamides were incorporated into phosphatidylcholine multilamellar and small unilamellar vesicles to measure their effects on the physical properties of membranes. X-ray diffraction analysis of the multilamellar vesicles revealed that the herbicides partitioned into the hydrocarbon chain region of the bilayer, but caused only minimal perturbations on hydrocarbon chain packing. 31P-NMR spectroscopy of these multilamellar vesicles showed both a broadening and lowering of the phase transition temperature of the bilayer lipids upon addition of the herbicides. 13C-NMR spectroscopy of small, unilamellar phosphatidylcholine vesicles was performed to measure the effects of the phenylamides on the chemical shifts and the spin-lattice relaxation times of the individual phosphatidylcholine carbon atoms. None of the added compounds had any measurable effect on the 13C-NMR chemical shifts of the phosphatidylcholine. However, the herbicides significantly modified spin-lattice relaxation times of certain of the lipid carbon atoms. These results generally indicate that the herbicides orient in the lipid bilayers such that the hydrocarbon chains of the phenylamides associate with the hydrocarbon chains of the lipid, whereas the phenyl moiety resides in the polar region of the bilayer.     

High-frequency ultrasonic absorption spectroscopy on aqueous suspensions of phospholipid bilayer vesicles

Between 1 MHz and 3 GHz the ultrasonic absorption coefficient has been precisely measured as a function of frequency for some aqueous suspensions of single-walled phospholipid bilayer vesicles. All solutions of the specially purified phospholipids clearly show excess absorption, reflecting three molecular relaxation processes with discrete relaxation times. Typical values for these times are 50, 3 and 0.5 ns. The attempt is made to relate these relaxation processes to mechanisms of rotational isomerization in the hydrocarbon chains. Some other molecular mechanisms which could also contribute to the ultrasonic excess absorption spectra are also briefly discussed.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)