The effects of A23187 on the phospholipid phase transition of large unilamellar vesicles (LUVs) as detected by ultrasound spectroscopy

The effect of the hydrophobic Ca2+ ionophore, A23187, on the phospholipid dynamics of large unilamellar vesicle (LUVs: 4: 1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG] membranes, as a function of A23187 content, was investigated using techniques sensitive to the phospholipid phase transition. The ultrasonic absorption per wavelength, alpha lambda, was determined with a double crystal acoustic interferometer, as a function of temperature and frequency for LUVs in the vicinity of their phospholipid phase transition. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) were also employed to probe the thermodynamics and molecular environment of the hydrocarbon side chains. With increasing A23187 content, the phase transition temperature (Tm) of the LUV suspensions remained near 42.0 degrees C, while the amplitude of alpha lambda at the phase transition increased dramatically. At Tm the relaxation frequency, where alpha lambda max occurs, decreased with A23187 content, suggesting that the relaxation rate of the event responsible for the absorption of ultrasound decreased. The ESR studies showed no change in the fluidity of the bilayer with the inclusion of 2 and 5 mol% A23187 in the C-12 region of the bilayer. Therefore, A23187 in LUV membranes slows the structural relaxation of the hydrocarbon side chains of the phospholipid bilayer at the phase transition.     

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.     

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.     

Interaction of small molecules with phospholipid bilayer membranes: permeability studies

The passage of a phospholipid through the gel to liquid crystal phase transition is associated with an increase in the motional freedom of its fatty acyl chains as measured by spectroscopic techniques and an essentially isothermal absorption of heat as measured by differential scanning calorimetry (DSC). In addition, bilayers formed from that phospholipid display a permeability maximum for both non-electrolytes and electrolytes in the temperature region of the phase transition. In this study the sodium (and in some cases glucose) permeabilities of liposomes composed of either dimyristoyl or dipalmitoyl phosphatidylcholine plus dicetylphosphate were measured in the presence of a group of benzene and adamantane derivatives known to increase fatty acyl chain motion below the lipid transition temperature (T_c) and in the case of the adamantanes to also lower the T_c as measured by DSC. None of these compounds change the temperature at which the permeability maximum occurs despite their lowering of the phospholipid T_c. That is, in the presence of these additives there is observed an apparent dissociation between the phase transition and the permeability maximum. It is proposed that the permeability maximum normally observed in the temperature region of the T_c is associated with the completion of the ‘melting’ process. Hence a compound could cause early ‘melting’ of the bilayer but not change its permeability properties if the temperature at which the ‘melting’ process neared completion was not changed.     

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.     

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.     

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.     

Fourier-transform infrared studies of CaATPase/phospholipid interaction: survey of lipid classes

CaATPase from rabbit skeletal muscle has been isolated, purified, delipidated, and reconstituted with retention of ATPase activity into lipid vesicles consisting respectively of 1,2-dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), and egg sphingomyelin. The effect of the enzyme on phospholipid order and melting characteristics were determined with Fourier-transform infrared spectroscopy. Taken together with prior data from this laboratory for 1,2-dipalmitoylphosphatidylcholine and 1,2-dioleoylphosphatidylcholine (DOPC), as well as for native sarcoplasmic reticulum (SR), three types of lipid response to protein incorporation have been observed: (1) Phospholipids with high levels of acyl chain unsaturation (DOPC or native SR) have their lipid acyl chains slightly ordered by CaATPase incorporation. The effect of protein on the gel-liquid crystal phase transition cannot be easily determined, since the cooperative melting even in these systems occurs at temperature well below 0 degrees C. (2) Phospholipids with saturated acyl chains show slightly lowered melting temperatures and reduced cooperativity of melting upon CaATPase insertion. In addition, protein induces (at most) slight disorder into the acyl chains at temperatures removed from the lipid melting point. (3) The strongest response is observed for phospholipids containing one saturated and one unsaturated chain (POPE or SOPC) or heterogeneous systems with low levels of unsaturation (egg sphingomyelin). In these cases, relatively low protein levels diminish the magnitude of or completely abolish the phospholipid phase transition. In addition, substantial disorder is introduced into the acyl chain compared with the pure lipid both above and below its transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase transitions of phospholipid single-wall vesicles and multilayers. Measurement by vibrational raman spectroscopic frequency differences

Raman spectroscopic frequency differences between selected carbon-carbon stretching modes of lipid hydrocarbon chains were determined as a function of temperature for use in monitoring lipid phase transition behavior and acyl chain disorder in both multilamellar and single-wall vesicles. Transition temperatues detected by this procedure for pure dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine multilayers were observed at $\text{39±1 °}\text{C}$ and $\text{23±1 °}\text{C}$, respectively. Although the phase transition for unilamellar vesicles of dipalmitoyl phosphatidylcholine occurred at nearly the same temperature as the multilayers, the crystal-liquid crystalline transition for the single-shell vesicles appeared to span a slightly broader temperature range, a characteristic consistent with irregularities in the packing arrangement of the hydrocarbon chains. Within the precision of the Raman spectroscopic method, however, the temperature behavior of both the multilamellar and the unilamellar dimyristoyl phosphatidylcholine assemblies appeared nearly identical. The temperature profile for the Raman frequency differences of an excess water sonicate of 25 mol percent cholesterol in dipalmitoyl phosphatidylcholine served as an example of the effect upon lipid phase transition characteristics of a bilayer component intercalated between the acyl chains. For this particular cholesterol-lipid system the phase transition was broadened over a 30 °C temperature range, in contrast to the narrow 5?4 °C range observed for pure multilayer and single-shell vesicle particles.     

A theoretical model of the temperature- and pressure-induced phase transition of phospholipid bilayers

A statistical thermodynamic model of phospholipid bilayers is developed. In the model, a new concept of a closely packed system is applied, i.e., a system of hard cylinders of equal radii, the radius being a function of the average number of gauche rotations in a hydrocarbon chain. Using this concept of a closely packed system, reasonable values are obtained for the change in specific volume at the order-disorder transition of lecithin bilayers. In addition to interactions between the lipid matrix and water molecules, between the head groups themselves and between hydrocarbon chains, as well as the intramolecular energy associated with chain conformation, the Hamiltonian of the membrane also includes the energy of the pressure field. Thus, the phase transition of phospholipid membranes induced not only by temperature hut also by hydrostatic pressure is described by this model simultaneously. In accordance with the experimental results, a linear relationship is obtained between the phase transition temperature and phase transition pressure. The other calculated phase transition properties of lecithin homologues. e.g., changes in enthalpy, surface area. thickness and gauche number per chain are in agreement with the available experimental data. The ratio of kink to interstitial conduction of bilayers is also estimated.     

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)     

The condensing effect of glucagon on phospholipid bilayers

Glucagon forms discoidal particles with dimyristoylphosphatidylcholine at temperatures below the phase transition. Under these conditions and at a lipid to protein molar ratio of 20 : 1, glucagon is observed to induce a closer packing of the phospholipid bilayer. Similar effects are observed upon the interaction of glucagon with dilauroylphosphatidylcholine. In the region of the phase transition the discoidal particles are observed by freeze-fracture electron microscopy to undergo end-to-end association leading to the formation of multilamellar structures containing only a few layers and having a large internal volume. Above the phase transition temperature the properties of the lipid appear to be unperturbed by glucagon according to either freeze-fracture or densitometer studies. These results further support the importance of phospholipid phase transitions in peptide-lipid interactions.     

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.     

Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study

Polarized Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy was applied to study the orientation of the linear pentadecapeptide antibiotic gramicidin D incorporated into phospholipid multibilayers, which were cast on a germanium ATR plate from chloroform solution. In DMPC and DPPC multibilayers, the CH2 stretching bands of lipid hydrocarbon chains were slightly shifted to the higher frequency side and bandwidth was increased in the presence of gramicidin. However, in DPPE multibilayers, frequencies and bandwidths of these bands were unaltered. In each case, gramicidin produced little effect on the orientation of lipid hydrocarbon chains, suggesting that gramicidin penetrates into lipid layers without noticeable perturbations. Upon incubation of cast films in contact with water above the gel-liquid-crystalline transition temperature (Tc) of lipids, the reorientation of gramicidin in lipid multibilayers occurred, the degree thereof depending upon the fluidity of the lipid hydrocarbon chains and the amount of surrounding water. In DMPC multibilayers, the helix axis of gramicidin was oriented almost parallel to the lipid hydrocarbon chains after incubation. In DPPC multibilayers, on the other hand, the helix axis of gramicidin was tilted on average about 15 degrees from the lipid hydrocarbon chains after incubation. However, in DPPE multibilayers, which are known to have the most rigid bilayer structures, the reorientation of gramicidin could not be seen.     

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe.

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Comparison of the orientational order of lipid chains in the L alpha and HII phases

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (L alpha) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the L alpha phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L alpha to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L alpha to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HII phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The molecular basis of mesomorphic phase transitions in phospholipid systems

The demonstrated existence and possible physiological relevance of mesomorphic phase transitions in cellular membranes suggests that a theoretical understanding of lipid phase behavior is biologically relevant. As a step in this direction, the gel to liquid crystal phase transition of phospholipid bilayers is examined. A qualitative mechanism involving configurational coupling of the lipid hydrocarbon chains is proposed to explain the transition. The predictions of the mechanism which pertain to the structure of the liquid crystal are explored and found to be in accord with the present experimental view of this phase.     

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.