Interactions of helical polypepetide segments which span the hydrocarbon region of lipid bilayers. Studies of the gramicidin A lipid-water system.

The polypeptide gramicidin A in a dimeric form is considered to form a helical structure which spans the hydrocarbon region of lipid bilayers. In the present investigation it is used as a model for the interactions of the polypeptide segments of transmembrane proteins within the hydrocarbon region of the lipid bilayers of biomembrane structures. A variety of physical techniques (X-ray diffraction, differential scanning calorimetry, optical and electron microscopy, Raman and electron spin resonance spectroscopy) are applied to a study of the interactions of this polypeptide within the phospholipid bilayers of dimyristoyl and dipalmitoyl lecithins in water, at temperatures both above and below the main endothermic phase transition of the pure lipids.Above the transition temperature of the lipid, the Raman studies show that the polypeptide perturbs the fluid lipid environment and causes a marked decrease in the number of gauche isomers of the lipid hydrocarbon chains, even at quite low relative molar concentrations of the polypeptide to lipid (1:150). At concentrations of phospholipid to polypeptide of less than 5:1, the electron spin resonance studies show the existence of two lipid regions within the bilayer. One region corresponds to the relatively fluid lipid region normally observed at these temperatures and the other to a relatively rigid lipid region. The latter is considered to arise from clusters of the polypeptide in which some of the lipid is entrapped.Below the lipid phase transition temperature, the pretransition endotherm observed with pure lipid-water systems is removed by small molar concentrations of the polypeptide (1:50) and the rippled appearance observed in freeze-fracture electron micrographs with pure dimyristoyl lecithin-water dispersions is replaced by a smooth appearance.The main lipid phase transition becomes broadened by the presence of increasing amounts of the polypeptide within the lipid bilayer as indicated by calorimetry, and electron spin resonance spectroscopy. The enthalpy of the lipid transition decreases linearly with increasing amounts of the polypeptide until, with dipalmitoyl lecithin, a concentration of approximately 20 lipids per polypeptide is reached. This is considered to correspond to the onset of an aggregation process which produces localised polypeptide-lipid clusters within the plane of the membrane.At concentrations of polypeptide less than five lipids per polypeptide, freezefracture electron microscopy shows the presence of liposomes with smooth fracture faces. At higher polypeptide concentrations, sheet-like structures are observed with smooth fracture faces.When a mixed lipid-water system (dilauroyl and dipalmitoyl lecithin) containing low concentrations of the polypeptide is slowly cooled, the calorimetric evidence shows that the polypeptide moves preferentially into the lower melting region of the bilayer, whereas at higher polypcptide eoncentrations a mixing of the two lipids takes place.The various results are discussed to provide insight pertinent to the organisation, interactions, aggregation properties, boundary layer and packing arrangements of helical polypeptides and proteins in reconstituted systems and natural biomembranes.     

Fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1.3-di(2-pyrenyl)propane

Intramolecular excimer formation of 1,3-di(2-pyrenyl)propane was used to study the fluidity of liposomes prepared from membrane polar lipids of Bacillus stearothermophilus. On the basis of spectral data, local polarity and polarizability parameters were established suggesting that the probe molecules are located well inside the membranes, but displaced towards the polar head groups of the phospholipid molecules. The excimerization rate is very sensitive to lipid phase transitions and pretransitions of synthetic pure lipid bilayers. In bacterial lipids from cultures grown at 55 and 68 degrees C, thermal profiles of excimer to monomer intensity ratios (I'/I) show a broad transition which is displaced to higher temperatures in response to the increase of the growth temperature; these results correlate well with differential scanning calorimetry data and fluorescence polarization of diphenylhexatriene. Additionally, lipid bilayers of bacteria grown at 68 degrees C exhibit a decreased membrane fluidity, as monitored by both fluorescent probes.     

Sublytic and lytic effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethylammonio)-1-propanesulfonate on phosphatidylcholine liposomes

The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 mM phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 mM surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 mM phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the Tc gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a "shoulder" appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.     

Enigmatic thermotropic phase behavior of highly asymmetric mixed-chain phosphatidylcholines that form mixed-interdigitated gel phases.

Twelve saturated mixed-chain phosphatidylcholines have been identified for which the thermotropic phase behavior observed upon cooling from the L alpha phase is dependent upon the thermal history of the sample in the gel phase. If fully hydrated samples of these lipids are cooled and soon thereafter examined by differential scanning calorimetry, one observes a single highly cooperative endotherm (the chain-melting phase transition) upon heating, and on subsequent cooling, a single exotherm that may occur at temperatures as much as 4-6 degrees C below that of the single endotherm observed upon heating. In contrast, if the samples are incubated in the gel state at low temperatures for prolonged periods of time, one observes a single heating endotherm as before, but two sharp exotherms upon cooling. The latter transitions occur at temperatures close to that of the single endotherm observed upon heating and the single cooling exotherm observed prior to incubation in the gel state. The combined enthalpy of the two cooling exotherms is the same as that of the single heating endotherm or the single cooling exotherm initially observed. Infrared spectroscopic and X-ray diffraction studies indicate that the structural conversions characteristic of liquid-crystalline/gel phase transitions occur at both of those cooling exotherms. Of the 12 lipids that exhibit this unusual behavior, nine fulfill the previously defined structural requirements for the formation of the so-called mixed-interdigitated gel phase, and there is evidence in the literature that one of the three remaining lipids also forms such a structure. Infrared spectroscopic studies of the other two lipids indicate that their gel phases exhibit spectroscopic features that closely resemble those of lipids that meet the previously defined structural criteria for the formation of mixed-interdigitated gel phases and that differ markedly from those of both saturated symmetric-chain and saturated mixed-chain phosphatidylcholines that do not normally form mixed-interdigitated gel phases. Also, electron density reconstructions based on small-angle X-ray diffraction studies of the gel phases of those two lipids indicate that the thickness of their gel phase bilayers is consistent with their forming mixed-interdigitated gel phases. Thus the unusual thermotropic phase behavior described here may be a general characteristic of phosphatidylcholines that form mixed-interdigitated gel phases. This unusual behavior is not associated with any major change in any of several physical properties of these lipid bilayers but may arise from an alteration of the size and/or structure of microdomains present in the liquid-crystalline phase.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Perturbation of phospholipid membranes by juvenile hormone

The effects of juvenile hormone and its analogs Altozar 4E and ZR-777 5E on the phase properties of liposomes prepared from dipalmitoyl phosphatidyl-choline (DPPC) have been examined by differential scanning calorimetry. Each of these compounds reduced the co-operativity of the gel to liquid-crystalline phase transition, which is manifest as a distinct broadening of the main transition endotherm, and split the transition into two distinguishable components centered at 34 and 37°C. However, there was no significant change in enthalpy of the main phase transition, suggesting that juvenile hormone and its analogs perturb the bilayer primarily in the vicinity of the phospholipid headgroups. Moreover, this perturbation does not appear to influence bilayer permeability since the osmotic swelling rates of liposomes prepared from either phosphatidylcholine or dipalmitoyl phosphatidylcholine that contained up to 33 mol% juvenile hormone were not significantly different from the swelling rates of corresponding liposomes containing no juvenile hormone. Splitting of the transition endotherm into two peaks appeared to be peculiar to compounds possessing juvenile hormone activity. A mixture of fatty acid methyl esters broadened the main transition of DPPC, but did not split the endotherm or shift the transition midpoint, and the insect hormone ecdysone had no discernible effect on the DPPC transition apart from eliminating the pretransition. The data indicate that juvenile hormone and its analogs can modulate the physical properties of phospholipid bilayers, and raise the prospect that some of the physiological effects of this hormone may be achieved through its influence on the molecular organization of membrane lipid.     

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.     

Synthesis, structure, and thermal properties of 1,2-dipalmitoylgalloylglycerol (DPGG), a novel self-adhering lipid

A novel diacyl glycerol-based lipid with a polyphenolic head group has been synthesized and characterized. X-ray diffraction experiments show that this lipid, 1,2-dipalmitoylgalloylglycerol (DPGG), hydrates to form gel phase bilayers at 20 degrees C with extremely narrow interbilayer fluid separations, indicating that apposing DPGG bilayers strongly adhere to each other. Differential scanning calorimetry shows that fully hydrated DPGG exhibits a pretransition exotherm (3.7 kcal/mol) at 52 degrees C and a high enthalpy (11.3 kcal/mol) main endothermic transition at 69 degrees C. These thermal properties are similar to those of galactosylceramides with similar hydrocarbon chain compositions. The adhesive and thermal properties of DPGG are likely due to both intermolecular hydrogen-bonding and hydrophobic interactions between the aromatic rings on the gallic acids.     

Physical studies on membrane lipids of Bacillus stearothermophilus temperature and calcium effects

Bacillus stearothermophilus was grown at the optimal temperature range (center, 65 degrees C), below it (48 and 55 degrees C), and above it (68 degrees C), in a complex medium with or without 2.5 mM Ca2+. The Ca(2+)-supplement improves growth at sub- and supraoptimal temperatures and extends it to higher temperatures (Jurado et al. (1987) J. Gen. Microbiol. 133, 507-513). The phospholipid composition of cultures obtained in the different growth conditions was studied. Phosphatidylethanolamine was always the major phospholipid (40 to 50% of the total phospholipid). Diphosphatidylglycerol, phosphatidylglycerol, a phosphoglycolipid (pgl) and two minor phospholipids (not identified) were also found in the polar lipid extract. The pgl shows a threefold concentration increase as the growth temperature raises from 48 to 68 degrees C. The thermotropic behavior of membrane lipids was studied by differential scanning calorimetry (DSC) and by means of two fluorescent probes of fluidity, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(2-pyrenyl)propane (2Py(3)2Py). The results reveal similar features and clearly show a shift of the temperature range of the phase transition to higher values and an increased structural order of the bilayer, as the growth temperature rises from 55 to 68 degrees C, but an opposite effect was observed from 48 to 55 degrees C. Although the Ca(2+)-supplement to the growth medium has no detectable effect, the addition of Ca2+ to the buffer of liposomes (Ca(2+)-liposomes) has a significant ordering effect at all growth temperatures. These liposomes show a shift of the transition range to higher temperatures and the fluorescent parameters (DPH polarization and intramolecular excimerization of the 2Py(3)2Py) detected an order increase of the probes environment, along and above the main phase transition. Spectra of 31P-NMR and polarized light microscopy clearly show that the lipid extracts exhibit, in all the conditions, typical lamellar phase geometry. We concluded that B. stearothermophilus controls the membrane lipid composition to compensate for the destabilizing effect of high temperatures on the membrane organization or to provide an appropriate packing of phospholipid molecules in a stable bilayer. At high temperatures, Ca(2+)-stimulatory effect on growth is presumably due to a direct Ca2+ interaction with the membrane phospholipids, inducing an increased structural order on the bilayer. The increase of the phase transition temperature in the total lipid extracts as compared with the respective polar lipid fractions probably indicates a stabilizing effect of neutral lipids on membrane bilayers.     

Phospholipid phase transitions in homogeneous nanometer scale bilayer discs

Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan fluorescence and differential scanning calorimetry. Laurdan is a fluorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity profile can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Differential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3-4 degrees C higher for both lipids when incorporated into Nanodiscs. These findings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins.     

Intermolecular interactions of lysobisphosphatidic acid with phosphatidylcholine in mixed bilayers

Lysobisphosphatidic acid (LBPA) can be regarded to represent a unique derivative of phosphatidylglycerol. This lipid is highly enriched in late endosomes where it can comprise up to 10-15 mol% of all lipids and in these membranes, LBPA appears to be segregated into microdomains. We studied the thermotropic behavior of pure dioleoyl-LBPA mono- and bilayers using Langmuir-lipid monolayers, electron microscopy, differential scanning calorimetry (DSC), and fluorescence spectroscopy. LBPA formed metastable, liquid-expanded monolayers at an air/buffer interface, and its compression isotherms lacked any indication for structural phase transitions. Neat LBPA formed multilamellar vesicles with no structural transitions or phase transitions between 10 and 80 degrees C at a pH range of 3.0-7.4. We then proceeded to study mixed LBPA/dipalmitoylphosphatidylcholine (DPPC) bilayers by DSC and fluorescence spectroscopy. Incorporating increasing amounts of LBPA (up to X(LBPA) (molar fraction)=0.10) decreased the co-operativity of the main transition for DPPC, and a decrease in the main phase transition as well as pretransition temperature of DPPC was observed yet with no effect on the enthalpy of this transition. In keeping with the DSC data for DPPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/LBPA mixed bilayers were more fluid, and no evidence for lateral phase segregation was observed. These results were confirmed using fluorescence microscopy of Langmuir-lipid films composed of POPC and LBPA up to X(LBPA)=0.50 with no evidence for lateral phase separation. As late endosomes are eminently acidic, we examined the effect of lowering pH on lateral organization of mixed PC/LBPA bilayers by DSC and fluorescence spectroscopy. Even at pH 3.0, we find no evidence of LBPA-induced microdomain formation at LBPA contents found in cellular organelles.     

On two-dimensional passive random walk in lipid bilayers and fluid pathways in biomembranes

Summary The lateral mobility of pyrene, pyrene decanoic acid, and 1-palmitoyl-2-pyrene decanoyl-phosphatidyl choline (pyrene lecithin) in lipid bilayers is determined by the excimer formation technique. This method is applied to vesicles of lecithins differing in chain length and in the degree of saturation of the hydrocarbon chains. These values are compared with results in cephalins of different chain length and in dipalmitoyl phosphatidic acid at variable pH. The influence of cholesterol is investigated. The results are analyzed in terms of the Montroll model of two-dimensional random walk. The jump frequency of the probe molecule within the lipid lattice is obtained. The advantage of this measure of transport in lipid layers is that it does not involve lipid lattice parameters.The main results of the present work are: (i) The lateral mobility of a given solute molecule in lamellae of saturated lecithins is independent of hydrocarbon chain length and rather a universal function of temperature. (ii) In unsaturated dioleyl lecithin the amphiphatic molecules have lateral mobilities of the same size as in saturated lipids. The jump frequency of pyrene, however, is by a factor of two larger in the unsaturated lecithin. (iii) The jump frequencies in phosphatidyl ethanolamines are about equal to those in lecithins. (iv) In phosphatidic acid layers the hopping frequencies depend on the chargers of the head groups of both the lipids and the probes. (v) Cholesterol strongly reduces the jump frequency in fluid layers. (vi) The lateral mobility in biological membranes is comparable to that in artificial lipid bilayers.The experimental results are discussed in terms of the free volume model of diffusion in fluids. Good agreement with the predictions made from this model is found. A striking result is the observation of a tilt in dioleyl-lecithin bilayer membranes from the hopping frequencies of pyrene and pyrene lecithin. A tilt angle of -17° is estimated.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Thermotropic behavior of liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and their mixtures

The thermotropic properties of multilamellar liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and several mixtures of these two lipids were studied with the application of excimer--forming optical probe pyrene and microcalorimetry. It was discovered that when the proportion of the egg yolk lecithin in the lipid mixture was raised the temperature of the main phase transition reduced. For all this, independent of the lipid mixture composition when the temperature was raised, apparently, polarity of pyrene microenvironment in the liposomes bilayers decreased. On the basis of the analysis of solidus and liquidus curves obtained from calorimetric studies of the lipid mixtures and bend points of Arrhenius anamorphose obtained during the pyrene excimer formation measurements some conclusions were made about the role of unmodified and hydrogenized egg yolk lecithin cluster formation in the determination of thermotropic properties of the liposomes from the above two lipids mixtures. High temperature phase transition discovered for the egg yolk lecithin while measuring the pyrene excimer formation is proposed to be closely connected with temperature-dependent changes in the organization of phospholipid heads on the interphase bilayer/H2O solution.     

Interaction of alamethicin with ether-linked phospholipid bilayers: oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry studies

The arrangement of the antimicrobial peptide alamethicin was studied by oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry in ether-linked phospholipid bilayers composed of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC). The measurements were performed as a function of alamethicin concentration relative to the lipid concentration, and results were compared to those reported in the literature for ester-linked phospholipid bilayers. At ambient temperature, alamethicin incorporates into the hydrophobic core of DHPC bilayers but results in more lipid disorder than observed for ester-linked 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers. This orientational disorder appears to depend on lipid properties such as bilayer thickness. Moreover, the results suggest that alamethicin inserts into the hydrophobic core of the bilayers (at high peptide concentration) for both ether- and ester-linked lipids but using a different mechanism, namely toroidal for DHPC and barrel-stave for POPC.     

Search for an endotherm in chloroplast lamellar membranes associated with chilling-inhibition of photosynthesis

The phase transition of chloroplast lamellar membrane lipids has been proposed to be the underlying cause of chilling-induced inhibition of photosynthesis in sensitive plants. Differential scanning calorimetry has been used to search for any endotherms arising from lipid state changes in chloroplast lamellar membranes of the chilling-sensitive plants cantaloupe , kidney bean, domestic tomato, and soybean. For comparison, calorimetric scans of chloroplast lamellar membranes from the chilling-insensitive plants spinach, pea, and wild tomato were made. A large reversible endotherm, extending from below 10 degrees to nearly 40 degrees C, was observed in chloroplast membranes from tomatoes of both chilling-sensitive (Lycopersicon esculentum Mill. cv. Floramerica ) and chilling-insensitive (L. hirsutum LA 1361) species. A much smaller endotherm, approximately 5 to 10% of the area of that seen in the two tomato species, and extending over a similar temperature range, was detected in chloroplasts from chilling-insensitive spinach and peas, and also was generally observed in chloroplasts from chilling-sensitive cantaloupe , kidney bean, and soybean. The enthalpy of these smaller endotherms indicates that, if the endotherm arose entirely from a lipid transition, then it corresponded to the melting of less than about 10% of the total membrane polar lipid. On the basis of these data it is concluded that there is no correlation between chilling sensitivity of photosynthesis and the presence or absence of a phase transition of bulk membrane lipids of the chloroplast lamellar membrane at temperatures above 5 degrees C.