Thermotropic properties of phospholipid analogues

To understand the structural bases for the polymorphism of phospholipids, it is often essential to study the properties of "unnatural" phospholipid analogues with modified polar headgroups and or backbone structures. While the thermodynamic characteristics of the "classical" hydrated-gel-to-liquid-crystalline phase transition often appear surprisingly insensitive to these aspects of phospholipid structure, the rich and diverse solid-phase polymorphism of phospholipids is in fact exquisitely sensitive to the nature of both the polar headgroup and the backbone moieties. The tendencies of different phospholipids to form nonlamellar phases at higher temperatures also depend strongly (and in a sometimes surprising manner) on fine details of the headgroup and backbone structures. These points are illustrated by discussions of how the structures of headgroup- and backbone-modified phospholipid analogues influence their proclivities to form distinct types of hydrated solid phases, dehydrated "crystralline" phases and nonlamellar phases.     

Thermotropic properties of saturated mixed acyl phosphatidylethanolamines

The mixed acyl phosphatidylethanolamine (PE) series C(18)C(18)PE, C(18)C(16)PE, C(18)C(14)PE, C(18)C(12)PE, and C(18)C(10)PE has been prepared from the corresponding phosphatidylcholines by phospholipase D mediated transphosphatidylation. The thermotropic behavior of unhydrated and hydrated preparations of these PEs has been investigated by differential scanning calorimetry and 31P NMR spectroscopy. Unhydrated preparations of the PEs undergo crystalline to liquid-crystalline transitions (Tm+h), which correspond to the simultaneous hydration and acyl chain melting of poorly hydrated crystalline samples. Hydrated preparations of the PEs undergo gel to liquid-crystalline transitions (Tm) when scanned immediately subsequent to cooling from temperatures above their respective Tm+hs. Multilamellar bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE pack without significant interdigitation of the phospholipid acyl chains across the bilayer center in the gel phase. C(18)C(10)PE multilamellar preparations exhibit a mixed-interdigitated gel phase packing of the phospholipid acyl chains. Hydrated bilayers of C(18)C(12)PE adopt a mixed-interdigitated gel phase packing at temperatures below 13.9 degrees C. Between 13.9 degrees C and the gel to liquid-crystalline transition temperature of 36.9 degrees C, the C(18)C(12)PE bilayer adopts a noninterdigitated gel phase packing. The metastable behavior of fully hydrated and partially hydrated preparations of the mixed acyl PEs has been investigated. Bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE exhibited little or no tendency toward regeneration of the crystalline phase. In contrast, bilayers of C(18C(12)PE and C(18)C(10)PE exhibited a metastability of the liquid-crystalline phase in the temperature interval between Tm and Tm+h, which can allow for the regeneration of the crystalline phase under certain conditions.Bilayers of C(18)C(12)PE exhibited an additional metastability of the noninterdigitated gel phase.     

Molecular mechanics and calorimetric studies of phosphatidylethanols

Phosphatidylethanols (PEths) are negatively charged diacyl phospholipids that are ubiquitously present in humans under the condition of alcohol intoxication. These lipids, derived in vivo from other naturally occurring phospholipids such as phosphatidylcholines (PC) via transphosphatidylation reaction as catalyzed by phospholipase D in the presence of ethanol, are well known to affect many biochemical properties of the cell membranes in humans. In this communication, we applied the combined approach of molecular mechanics (MM) simulations and high-sensitivity differential scanning calorimetry (DSC) to investigate the structure and phase transition behavior of PEth. We first determined the energy-minimized structures of tetrameric C(15):C(15)PEth arranged in two types of packing motif by the MM approach. An inwardly bent orientation of the lipid headgroup was observed; specifically, the methyl terminus of PEth's headgroup was juxtaposed intramolecularly to the C(2) atom of the sn-2 acyl chain. Clearly, this headgroup conformation was rather unique among all naturally occurring phospholipids. Subsequently, the phase transition behavior of the fully hydrated lipid bilayers prepared individually from 11 species of saturated C(X):C(Y)PEth with the same MW was studied by DSC, and the resulting Tm values were codified in terms of the normalized acyl chain asymmetry (deltaC/CL). A V-shaped Tm profile was observed in the plot of Tm versus deltaC/CL for each subclass of these lipids, suggesting two types of packing motif for C(X):C(Y)PEth at T < Tm. Moreover, it was observed that within each packing motif these Tm values were, on average, 2.0 +/- 0.9 degrees C smaller than the Tm values of the corresponding saturated PC. However, based on the unique headgroup conformation of PEth, we were able to predict that monounsaturated PEth with a cis double bond near the H2O/hydrocarbon interface would exhibit a higher Tm than the corresponding PC. Most interestingly, this prediction was indeed borne out by DSC results obtained with C(18):C(20:1delta5)PC and C(18):C(20:1delta5)PEth.     

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides. Part II. Disaccharide headgroups

We have investigated the thermotropic and lyotropic properties of some long chain alkyl glycosides with disaccharide headgroups. The thermotropism was measured with polarising microscopy and additionally the lyotropism with the contact preparation method, Fourier-transform Infrared (FTIR) spectroscopy, X-ray diffraction and small angle neutron scattering. A broad thermotropic as well as lyotropic polymorphism was found. The compounds displayed thermotropic S(A) (lamellar) and cubic phases, and the investigation of the lyotropic phase behaviour led to the observation of inverted bicontinuous cubic V(II) phases, lamellar L(alpha) phases, normal bicontinuous cubic V(I) phases, normal columnar H(I) phases, normal discontinuous cubic I(I) phases and lyotropic cholesteric phases. The phases are discussed with respect to the chemical structures that have been varied systematically to derive structure-property relationships.     

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol.

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.     

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides. Part I: monosaccharide headgroups

A systematic structure variation of a classical amphiphile (dodecyl-beta-D-glucopyranoside) is performed, demonstrating the influence of anomeric linkage, configuration, ring size and flexibility as well as electric charges on the mesophase behaviour. In addition, we have investigated the thermotropic and lyotropic properties of some long chain alkyl glycosides with monosaccharide headgroups. The thermotropism was measured with polarizing microscopy and differential scanning calorimetry, and additionally the lyotropism with FTIR spectroscopy and X-ray diffraction.     

Thermotropic properties of human erythrocyte membrane proteins as affected by hydroxychloroaromatic compounds

The thermal stability of the anion transport protein (band 3) and other proteins of the human erythrocyte membrane, as influenced by hydroxychloroaromatic (HO-Cl2-Ar) compounds, was studied by differential scanning calorimetry. Various hydroxychlorodiphenyl ethers (HO-Clx-DPEs) and hexachlorophene, but not pentachlorophenol, caused a marked decrease in the thermal stability of band 3. Most of the other calorimetric transitions of the erythrocyte membrane were only slightly affected. The activity of HO-Clx-DPEs toward lowering the transition temperature of band 3 generally increased with the degree of chlorination, and was somewhat dependent on the position of hydroxyl substitution. At higher concentrations of HO-Clx-DPEs, there was a decrease in the enthalpy change and a broadening of the endothermic transition of band 3. The order of effectiveness of these compounds, as determined from band 3 denaturation temperatures, was similar to the order of potency previously observed for hemolysis of human erythrocytes.     

Thermotropic properties of dispersions of cholesterol with tetraether lipids from Thermoplasma acidophilum

The main glycophospholipid from Thermoplasma acidophilum is composed of a diisopranol-2,3-glycerotetraether. The fraction of pentane cyclizations of its hydrocarbon chains increases with the growth temperature of the source organism (39-59 degrees C). Hydrated mixtures of these lipids together with cholesterol have been studied by calorimetry. With the reduction of the phase transition temperatures and enthalpy changes of the transitions, cholesterol is readily incorporated into lipid monolayers in the liquid-crystalline and the (metastable) solid-analogue phase. Lipid samples with a high number of acyclic hydrocarbon chains form a stable and a metastable solid-analogue phase. With the increasing concentration of cholesterol the metastable solid-analogue phase is stabilized and the time constant for the formation of the stable solid-analogue phase is prolonged.     

Thermotropic lipid clustering in tetrahymena membranes.

The effect of temperature on the core structure of endoplasmic reticulum membranes has been visualized directly in cells of the poikilothermic eukaryote Tetrahymena pyriformis by freeze-etch electron microscopy. Moreover, the effect of temperature on the smooth microsomal membrane vesicles isolated from these cells, as well as on the extracted membrane lipids, has been examined by fluorescence probing, electron spin resonance, proton nuclear magnetic resonance, and calorimetry. Freeze-etch electron microscopy of T. pyriformis cells, equilibrated at different temperatures between 28 and 5 degrees, reveals the emergence of smooth areas on the fracture faces of endoplasmic reticulum membranes at temperatures below similar to 17 degrees. In this temperature range, we also find discontinuities in the glucose 6-phosphatase activity, in the fluorescence intensity of 8-anilino-1-naphthalensulfonate, in the partition of 4-doxyldecane, and in the separation of the outer hyperfine extrema of 5-doxylstearic acid in the microsomal membranes. These membranes apparently contain at least two lipid environments of different fluidity as indicated by the 12-doxylstearic acid spin-label. Proton nuclear magnetic resonance of the extracted membrane lipids indicates an abrupt change of the fatty acid chain mobilities at temperatures below similar to 17 degrees. This, however, is not due to a true thermal liquid crystalline in equilibrium crystalline phase transition. Calorimetric measurements also support this conclusion. The thermotropic alterations observed within the membranes are interpreted to be due primarily to a clustering of "rigid" liquid crystalline lipid environments which exclude membrane-intercalating proteins.     

Thermotropic properties of bipolar lipids of Sulfolobus solfataricus and of their mixtures with dipalmitoylphosphatidylcholine

The thermotropic properties of the bipolar lipids, glycerol dialkylglycerol tetraether (GDGT) and glycerol dialkylnonitol tetraether (GDNT), were determined at different degrees of hydration and in mixtures with dipalmitoylphosphatidylcholine (DPPC). The number of water molecules rendered unfreezable by the GDNT molecule is 10+/-1.5 and that by the GDGT molecule 2.8+/-0.7 or about 1.1-1.5 H2O molecules per OH group. Binding of water molecules causes randomization of the two polar heads from the oriented form prevailing in the dry state. The hydration seems to be a cooperative process extending over a whole lipid domain. DPPC added in small amounts to GDNT interacts preferentially with the nonitol halves of the molecules separating them from the glycerol half molecules. In the cooperative interaction domain each DPPC molecule is surrounded by up to six GDNT molecules. Cooperative domains formed during the interaction of DPPC with GDGT are less pronounced. In both cases they affect the thermotropic properties of the system.     

Thermotropic mesomorphism of cholesteryl myristate. An electron diffraction study

Electron diffraction measurements on heated or cooled microcrystals of cholesteryl myristate, which are grown from solution or epitaxially, on benzoic acid, provide further structural information about its mesomorphic behavior. At subambient temperatures (less than -65 degrees C), a new crystal form is observed which doubles the unit cell axes in the (001) plane. At the major crystalline in equilibrium with smectic endotherm at 70 degrees C, evidence is found for the existence of a pretransition crystal packing. The smectic phase, which coexists with this pretransition crystal form, is composed of relatively well-ordered layers, probably with a monolayer-type packing. Cooling the cholesteric phase to the crystalline form causes a rotational disorder which is not yet understood.     

Hydration pressure and phase transitions of phospholipids. II. Thermotropic approach

It is widely known that dehydration increases the main phase transition temperature of phospholipids. A mathematical analysis now shows that hydration pressure can be calculated by the dehydration-induced shift of the phase transition temperature. The hydration-dependent piezotropic and thermotropic phase transitions were determined by using calorimetry and FT-IR spectroscopy, and the application of our approach gives hydration pressure parameters that agree very well with the values obtained with the osmotic stress method.     

Molecular arrangements in sphingolipids. Thermotropic phase behaviour of tetracosanoylphytosphingosine

The phase behaviour of a ceramide species containing C₁₈-phytosphingosine and C₂₄-fatty acid was studied by X-ray diffraction methods, in order to elucidate the packing principles of lipids with unequally long hydrocarbon chains. Six solid phases were observed. In five of them, the ceramide molecules have an extended, V-shaped conformation and pack in single layer arrangements with the sphingosine and fatty acid chains forming separate matrices. Differences between these phases are mainly due to thermotropic changes in packing efficiency and thus in tilt of the hydrocarbon chains. The chain packing undergoes transitions from triclinic (T|) to monoclinic (M|) and hexagonal, and between orthorhombic (O⊥) and hexagonal subcell arrangements, respectively. Only one case was observed, in which the molecules pack with their two chains parallelly stacked in a double layer arrangement in which the long fatty acid tails deeply interdigitate between the two opposite layer halves. In a natural membrane containing different lipids, however, long fatty acid tails probably arrange randomly and contribute to the formation of a liquid hydrocarbon zone in the center of the bilayer.     

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides: part III: pH-sensitive headgroups

As part of a series of papers, the influence of carbohydrate headgroups and aliphatic chains on the mesogenic properties of glycolipids was investigated. Alkyl glycosides with different types of aliphatic chains were synthesised. Neutral glycolipids were oxidized to their uronic acid derivatives, using the well established TEMPO-oxidation. For comparison a 6-deoxy-6-amino alkylglucopyranoside was synthesised. In addition, the thermotropic and lyotropic phase behaviour of the synthesised compounds were investigated. The thermotropism was characterised by polarising microscopy, the lyotropism by the contact preparation method.     

Thermotropic behavior of fatty acid ethyl esters in phospholipid liposomes.

The thermotropic behavior of a series of synthetic fatty acyl ethylesters (FAEE) in multilamellar liposomes has been studied by differential scanning calorimetry and monitoring the changes in polarization emitted by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Their thermotropic behaviour has been compared to that of the homologous fatty acids (FA) from which they are synthesized in vivo in the presence of ethanol. Compared to the correspondent FA, saturated FAEE show, depending on the chain length, a minor rigidifying effect or even a slight fluidizing effect on phospholipid bilayers. Unsaturated FAEE show, compared to the homologous FA, slightly greater fluidizing properties. The difference between FA and FAEE is more evident in single component phospholipid liposomes in the gel phase, and in mixed liposomes of two lipids at temperatures at which microdomains of gel and liquid zones coexist. The calorimetric data suggest that FAEE are completely miscible with phospholipids both in the gel and liquid phases; they appear to destabilize the bilayer wherein the ethoxy head group interferes with the intrinsic phospholipid interactions.     

Thermotropic phase behavior of phosphatidylcholines with omega-tertiary-butyl fatty acyl chains.

The thermotropic phase behavior of a homologous series of phosphatidylcholines containing acyl chains with omega-tertiary butyl groups was studied by differential scanning calorimetry, Fourier transform infrared spectroscopy, and 31P-nuclear magnetic resonance spectroscopy (31P-NMR). Upon heating, aqueous dispersions of these lipids exhibit single transitions which have been identified as direct conversions from Lc-like gel phases to the liquid-crystalline state by both infrared and 31P-NMR spectroscopy. The calorimetric data indicate that the thermodynamic properties of the observed transition are strongly dependent upon whether the acyl chains contain an odd- or an even-number of carbon atoms. This property is manifest by a pronounced odd/even alternation in the transition temperatures and transition enthalpies of this homologous series of lipids, attributable to the fact that the odd-numbered compounds form gel phases that are more stable than those of their even-numbered counterparts. The spectroscopic data also suggest that unlike other lipids which exhibit the so-called odd/even effect, major odd/even discontinuities in the packing of the polymethylene chains are probably not the dominant factors responsible for the odd/even discontinuities exhibited by these lipids, because only subtle differences in the appropriate spectroscopic parameters were detected. Instead, the odd/even alternation in the physical properties of these lipids may be attributable to significant differences in the organization of the carbonyl ester interfacial regions of the lipid bilayer and to differences in the intermolecular interactions between the terminal t-butyl groups of the odd- and even-numbered homologues. Our results also suggest that the presence of the bulky t-butyl groups in the center of the lipid bilayer reduces the conformational disorder of the liquid-crystalline polymethylene chains, and promotes the formation of Lc-like gel phases. However, these Lc-like gel phases are considerably less ordered than those formed by saturated, straight-chain lipids.     

Thermotropic behavior of monoglucocerebroside--dipalmitoylphosphatidylcholine multilamellar liposomes

The thermotropic behavior of multilamellar liposomes prepared from mixtures of glucocerebroside and dipalmitoylphosphatidylcholine has been studied by high-sensitivity scanning calorimetry. It is shown that glucocerebroside has a marked effect on the gel--liquid crystalline transition of dipalmitoylphosphatidylcholine. The pretransition seen in pure samples of dipalmitoylphosphatidylcholine is undetectable at small mode fractions of glucocerebrosides (less than 10%). The main transition is shifted to higher temperatures and becomes broader and less cooperative in the presence of glucocerebroside. The enthalpy change of the main transition decreases with increasing the glucocerebroside content. However, this decrease is not linear with the glucocerebroside/phospholipid mole ratio. Glucocerebroside itself does not show a separate transition in the temperature range of these studies (10--75 degree C). The origin of these effects and their dependence on the glucocerebroside content suggest that the in-plane distribution of glucocerebroside molecules is affected by the physical state of the lipid bilayer and by the glucocerebroside/phospholipid mole ratio.     

Thermotropic phase behavior and stability of monosialoganglioside micelles in aqueous solution.

The thermotropic phase behavior of monosialoganglioside in a dilute aqueous dispersion at pH 6.8 was measured by using synchrotron radiation small-angle x-ray scattering and was analyzed by a shell-modeling method. Previous calorimetric studies on ganglioside systems have shown quite different thermotropic behaviors from other biological lipid systems, however, the details have still been ambiguous. Because of high statistical data and a shell-modeling analysis, we could elucidate the internal structural change of monosialoganglioside micelle induced by the elevation of temperature from 6 to 60 degrees C, that is, the shrinkage of the hydrophilic region and the slight expansion of the hydrophobic region occurring simultaneously, accompanying the elongation of the axial ratios of the ellipsoidal micelles. The model structures obtained explain the changes in the experimental scattering curves, the distance distribution functions, and the gyration radii. In addition we have also found an evident thermal hysteresis in the scattering curves and in the structural parameters. The present result suggests that the thickness of the hydrophilic region, namely, the conformation of oligosaccharide chains, is sensitive to a change of temperature.     

Thermotropic behavior of sphingophospholipids of marine invertebrates

Thermotropic behavior of total fraction of sphingomyelin (SM) and its forms, sphingomyelin that contains acyl residues of normal fatty acids (SMN) and sphingomyelin that is Nacylated predominantly with hydroxy acids (SMH) as well as of ceramideaminoethylphosphonate (CAEP) has been studied by the method of differential scanning microcalorimetry. It was shown that chromatographically pure anhydrous sphingophospholipids isolated from different species of marine invertebrates have no phasic transitions in the entire temperature interval of scanning, from-100 to 100°C, whereas partially hydrated samples or mixtures of sphingophospholipids with glycerophospholipids (phosphatidylcholine or phosphatidylethanolamine isolated from corresponding marine invertebrates had phasic transitions in a large temperature range with thermoabsorption maxima in the areas of -40–20°C and 20–80°C. It is suggested that sphingophospholipids, as compared to marine invertebrate glycerophospholipids, have a much lower capability for self-organization, and to form regular supramolecular sphingophospholipid structures both in artificial systems and in biomembranes, such orientants as glycerophospholipids and water are necessary. Possible causes of an unusual behavior of sphingophospholipids of marine invertebrates as compared to glycerophospholipids are discussed.