Comparative effects of melittin and its hydrophobic and hydrophilic fragments on bilayer organization by Raman spectroscopy.

For bilayer systems consisting of 1,2-dimyristoyl phosphatidylcholine (DMPC) incubated with melittin, a polypeptide capable of integrating itself within the membrane, temperature profiles derived from Raman spectroscopic data indicate the existence of an immobilized lipid annulus surrounding the polypeptide. In particular, temperature profiles derived from C--H, C--D and C--C stretching mode parameters for 25:1, 14:1 and 10:1 lipid:protein mole ratios exhibit two order-disorder transitions. The primary (lower) gel to liquid crystalline phase transition is depressed when polypeptide concentration is increased. The concentration-independent higher temperature transition is associated with a fluidization of the immobilized boundary lipids present at the lipid-polypeptide interface within the bilayer. We estimate that five to seven lipids are involved in this discrete boundary layer around the inserted membrane component. The behavior of the intrinsic hydrophobic (residues 1-19) and of the extrinsic hydrophilic (residues 20-26) portions of melittin in the bilayer is compared with the properties of the intact polypeptide. We emphasize evidence that both intrinsic and extrinsic components immobilize lipids contiguous to the polypeptide.     

Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.     

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.     

Vibrational raman spectra of lipid systems containing amphotericin B

Resonance-enhanced and normal vibrational Raman spectra were observed for both multilamellar and single-wall vesicle assemblies of dimyristoyl phosphatidylcholine containing amphotericin B, a channel-forming polyene antibiotic, and cholesterol. The decrease in the frequency of the polyene antibiotic C  C stretching mode at 1556 cm^?1 and the increase in intensity of the CCH in-plane deformation mode at 1002 cm^?1 indicate that amphotericin B is ordered in a lipid-cholesterol medium similarly to the solid, but is surrounded by a slightly more polar environment. The intensity of the C  C stretching mode I₁₅₅₆ decreases 4-fold during the broadened gel to liquid crystalline phase transition (16–32°C) of dimyristoyl lecithin-cholesterol (4 : 1) multilayers. Other resonance-enhanced vibrations of amphotericin B exhibit similar behavior. For amphotericin B in pure dimyristoyl lecithin multilayer or vesicle systems, however, the vibrational intensity associated with the C  C stretching mode remains constant during the melting of lipid hydrocarbon chains. In addition, a third effect occurs in liquid crystalline egg lecithin-cholesterol (4 : 1, mol ratio) multilayers in which I₁₅₅₆ first increases by 25% between 3 and 25°C, in parallel with the loss of active channels, and then remains constant as the temperature increases from 25 to 42°C. This latter intensity pattern is masked in the dimyristoyl lecithin-cholesterol system by the overwhelming effect upon the C  C mode from changes in the lipid chain packing characteristics which occur during the phase transition.The broadened phase transition in 4 : 1 dimyristoyl lecithin-cholesterol multilayers (16–32°C), as followed by the ratio of intensities at 2880 and 2850 cm^?1 (asymmetric and symmetric methylene C-H stretching modes, respectively) is slightly narrowed by the addition of amphotericin B, and effect from which a binding stoichiometry at 24° of 1 : 1 amphotericin B : cholesterol is estimated. This stoichiometry was confirmed by differential calorimetric scans, which also show the presence of a peak proportional to cholesterol content.Raman I_2880/2850 peak height ratios in pure dimyristoyl lecithin bilayers were increased over the 14–38°C range by amphotericin B, a spectral effect which suggests an ordering of the lipid matrix perhaps as a consequence of the polyene binding to the bilayer surface. For bilayers containing cholesterol, the ratios of intensities of the 2935 cm^?1 feature, composed mainly of acyl chain terminal methyl and underlying methylene C-H stretching modes, to the 2850 cm^?1 feature are significantly increased by amphotericin B. This effect indicates that the antibiotic penetrates the bilayer in the lipid-sterol system.     

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.     

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37°C without vesicle fusion.The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing ¹³C NMR using N-¹³CH_3? labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (halftime 4 h) in the temperature range at which the hydrocarbon phase transition occurs.The activation energy of the flip-flop rate above the phase transition is 23.7 ± 2.0 kcal/mol.     

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).     

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.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

The effect of exogenous glycerophospholipids on the fluorescence polarization ratios of Escherichia coli cells labelled with diphenylhexatriene

Saturated, unsaturated, and short acyl chain analogues of phosphatidylcholine and phosphatidylcholine and phosphatidylethanolamine were incorporated into a deep heptoseless mutant of Escherichia coli, strain D21F2, and into the parent wild-type strain, K12. Normal and lipid-treated cells or lipid extracts from such cells were labelled with diphenylhexatriene and their fluorescence polarization ratios were measured as a function of temperature. Incorporations of dipalmitoyl analogues of phosphatidylethanolamine and/or phosphatidylcholine in the presence of Ca²⁺ caused an increase in polarization ratios over a wide temperature range and the appearance of new phase transitions at 25–30°C as measured in whole D21F2 cells. Incorporation into D21F2 of the dioleoyl analogues of these glycerophospholipids under similar conditions had the opposite effect on the polarization ratios and, in the case of dioleoylphosphatidylethanolamine, caused the occurrence of a new phase transition at 20°C. Incorporation of these same lipids in K12 cells, in the presence of Ca²⁺, caused changes in the polarization ratios similar to those recorded for D21F2 cells when measurements were made on whole cells. Furthermore incorporation of didecanoyl-phosphatidylcholine in wild-type cells, in the presence of Ca²⁺, substantially decreased the polarization ratio and broadened the phase transition as could be measured with cell preparations. Since Ca²⁺ stimulates incorporation of lipid, the changes in polarization ratio were always greater when cells had been exposed to exogenous lipid in the presence of this cation. However, even in cells not treated with lipid, Ca²⁺ caused increases in the polarization ratio and affected the thermotropic structural transitions. The polarization ratios of extracted lipids were always reduced when compared to whole cells. Generally there was an attenuation of any differences in polarization ratio between normal and glycerophospholipid-treated samples. Extracted lipids also displayed broadened phase transitions. The results as a whole indicated that E. coli cells respond to the uptake of lipid and to the presence of Ca²⁺ by changes in their thermotropic mesomorphic behaviour. These changes reflect to a large extent the fluidity of the incorporated lipid and are exerted on a structural system the phase transitions of which are strongly influenced by the presence of non lipid components in the membrane.     

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23 degrees C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.     

Studies of thermotropic phospholipid phase transitions using scanning densitometry

The thermotropic phase transitions were determined for a variety of phospholipids including dimyristoyl (DMPC) and distearoyl phosphatidylcholine (DSPC); dimyristoyl (DMPE), dioleoyl (DOPE) and egg phospatidylethanolamine (PE); egg and bovine brain sphingomyelin (SM) and bovine brain phosphatidylserine (PS) in the presence and absence of calcium or magnesium. The gel to liquid crystal phase transition is accompanied by a 2–4% increase in volume for a variety of phospholipids. This transition can be readily detected by scanning densitometry with multilamellar suspensions of phospholipids. In contrast, the liquid crystal to hexagonal phase transition does not involve any detectable change in volume. In addition, the volume coefficient of expansion for the hexagonal phase is similar to that found for several other bilayer systems. PS in the presence of Ca²⁺, SMs and DMPC at 50°C all have lower values of the volume coefficient of expansion. This property may be correlated with the resistance of these systems to the formation of additional gauche isomers in the hydrocarbon chains with increasing temperatures resulting in lowered permeability.     

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

Molecular conformations of cerebrosides in bilayers determined by Raman spectroscopy.

Vibrational Raman spectra of the solid and gel phases of bovine brain cerebrosides and the component fractions, kerasin and phrenosin, provide conformational information for these glycosphingolipids in bilayer systems. The carbon-carbon stretching mode profiles (1,150-1,000 cm-1) indicate that at 22 degrees C the alkyl chains assume an almost all-trans arrangement. These spectral data, combined with those from the C-H stretching region (3,050-2,800 cm-1), show that phrenosin forms the most highly ordered polycrystalline solid and kerasin the most ordered gel phase. The conformation of the unsaturated, 24-carbon acyl chains is monitored independently by a skeletal stretching mode at 1,112 cm-1. The alkyl chains in the kerasin and phrenosin gels are sufficiently extended to allow interdigitation of the 24-carbon acyl chains across the midplane of the bilayer. The amide I vibrational mode occurs at a lower frequency in solid phrenosin than kerasin, a shift consistent with stronger hydrogen bounding. This band is broadened and shifted to higher frequencies, however, in the phrenosin gel phase. In both the solid and gel phases natural cerebroside exhibits a composite amide I mode. The disruptive effects on cerebroside chain packing and headgroup orientation arising from mixing with dimyristoyl phosphatidylcholine are examined. Vibrational data for cerebroside are also compared to those for ceramide, sphingosine, and distearoyl phosphatidylcholine structures. Spectral interpretations are discussed in terms of calorimetric and X-ray structural data.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

High sensitivity differential scanning calorimetry of the bilayer to hexagonal phase transitions of diacylphosphatidylethanolamines

The bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine has been detected for the first time by differential scanning calorimetry. The observed transition is dependent on scan rate. This dependence can be explained by assuming that at rapid scan rates, the rate of conversion of bilayer to hexagonal phase is too slow at low temperatures for equilibration to take place. At higher temperatures the rate of interconversion becomes more rapid. The transition is observed to occur at 14°C using a scan rate of 0.74 K/min while it is centered at 8°C using a scan rate of 0.19 K/min. The enthalpy of the transition is 290 ± 40 cal/mol lipid and the transition is characterized by a ΔCp of −9 ± 1 mcal K⁻¹ (g lipid)⁻¹. The bilayer to hexagonal phase transition of dielaidoylphosphatidylethanolamine and of 1-palmitoy1-2-oleoylphosphatidylethanolamine occurs at 65.6°C and 71.4°C, respecitvely, with a corresponding transition enthalpy of 450 ± 20 and 400 ± 30 cal/mol lipid. The transitions of these phosphatidylethanolamines, occuring at higher temperatures, are independent of scan rate and show a higher degree of cooperativity than that of dioleoylphosphatidylethanolamine. Compared with the gel to liquid-crystalline transition of bilayer phospholipids the transition to hexagonal phase has a much lower enthalpy.