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.     

IR spectroscopic determination of gel state miscibility in long-chain phosphatidylcholine mixtures.

We report on the gel-state microaggregation in binary mixtures of diacylphosphatidylcholines over temperatures ranging from -19 degrees C to near the gel-to-liquid crystal transition. Microaggregates with lateral dimensions in the range 1-100 chains were detected and measured with an isotope infrared method that relates the splitting or the shape of the methylene scissors band to aggregate size. Measurements were made on fully hydrated dispersions of diC18DPC/diC20HPC, diC18DPC/diC22HPC, and diC18DPC/diC24HPC at molar ratios of 4:1. Low levels of aggregation were determined with reference to the spectrum of the random mixture diC18DPC/diC18HPC. For diC18DPC/diC20HPC at -19 degrees C, which previous calorimetric measurements have indicated is a nearly ideal, we found about 4% of the minority component chains to be involved in aggregates. For diC18DPC/diC22HPC, the value increased to about 11%. DiC18DPC/diC24HPC was found to be highly fractionated, in agreement with the earlier studies. The unit subcell, which defines the type of acyl-chain packing, was determined for the components of the mixtures. The temperature behavior of the phases and the temperatures at which the minority component domains undergo dissolution were determined.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 1. Differential scanning calorimetric studies

The thermotropic phase behavior of aqueous dispersions of phosphatidylcholines containing one of a series of methyl iso-branched fatty acyl chains was studied by differential scanning calorimetry. These compounds exhibit a complex phase behavior on heating which includes two endothermic events, a gel/gel transition, involving a molecular packing rearrangement between two gel-state forms, and a gel/liquid-crystalline phase transition, involving the melting of the hydrocarbon chains. The gel to liquid-crystalline transition is a relatively fast, highly cooperative process which exhibits a lower transition temperature and enthalpy than do the chain-melting transitions of saturated straight-chain phosphatidylcholines of similar acyl chain length. In addition, the gel to liquid-crystalline phase transition temperature is relatively insensitive to the composition of the aqueous phase. In contrast, the gel/gel transition is a slow process of lower cooperativity than the gel/liquid-crystalline phase transition and is sensitive to the composition of the bulk aqueous phase. The gel/gel transitions of the methyl iso-branched phosphatidylcholines have very different thermodynamic properties and depend in a different way on hydrocarbon chain length than do either the "subtransitions" or the "pretransitions" observed with linear saturated phosphatidylcholines. The gel/gel and gel/liquid-crystalline transitions are apparently concomitant for the shorter chain iso-branched phosphatidylcholines but diverge on the temperature scale with increasing chain length, with a pronounced odd/even alternation of the characteristic temperatures of the gel/gel transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing dl-methyl anteisobranched fatty acids. 1. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of nine dl-methyl branched anteisoacylphosphatidylcholines was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The calorimetric studies demonstrate that these compounds all exhibit a complex phase behavior, consisting of at least two minor, low-enthalpy, gel-state transitions which occur at temperatures just prior to the onset of the gel/liquid-crystalline phase transition. In addition, at still lower temperatures, anteisobranched phosphatidylcholines containing fatty acyl chains with an odd number of carbon atoms show a major, higher enthalpy, gel-state transition, which was assigned to a conversion from a condensed to a more loosely packed gel phase. No such transition was observed for the even-numbered compounds in aqueous dispersion, but when dispersed in aqueous ethylene glycol, a major gel-state transition is clearly discernible for two of the even-numbered phospholipids. The major gel-state transition exhibits heating and cooling hysteresis and is fairly sensitive to the composition of the bulk aqueous phase. 31P NMR spectroscopic studies indicate that the major gel-state transition is accompanied by a considerable change in the mobility of the phosphate head group and that, at temperatures just prior to the onset of the gel/liquid-crystalline phase transition, the mobility of the phosphate head group is comparable to that normally exhibited by the liquid-crystalline state of most other phospholipids. The temperatures at which the gel/liquid-crystalline phase transition occurs and the enthalpy change associated with this process are considerably lower than those of the saturated n-acyl-PC's of comparable acyl chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of replacement of a double bond by a cyclopropane ring in phosphatidylethanolamines: a 2H NMR study of phase transitions and molecular organization

The thermotropic behavior and molecular properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine (PDSPE) have been investigated by 2H NMR spectroscopy using samples selectively labeled at the 5'-, 9'-, 10'-, and 16'-positions of the sn-2 chains. Comparison with the corresponding phosphocholine analogues (POPC and PDSPC), obtained as intermediate synthetic products, was used to monitor the role of the polar head group. Replacement of the choline moiety by ethanolamine increased the gel to liquid-crystal transition temperature by 10-32 degrees C and led to a significantly higher ordering of the fatty acyl chains in the liquid-crystalline bilayer state. The lateral compression effect, due to the smaller area per polar head group in PE, results in a bilayer to hexagonal phase transition at elevated temperatures. The effects on both PC and PE due to replacement of the olefinic group by a cyclopropane unit are similar. A decrease in the temperature of the gel to liquid-crystal phase transition, Tc, is observed upon introduction of a cyclopropane ring; it goes from 26 degrees C in POPE to approximately 10 degrees C in PDSPE. In addition, a very significant broadening of the transition profile is observed. These observations are consistent with the poor packing ability of mixed saturated and cyclopropane-containing chains due to the bulky substituent effect. The temperature of the bilayer-hexagonal phase transition of PE samples was decreased by 15-20 degrees C on replacement of oleoyl chains by dihydrosterculoyl chains at the sn-2 position.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Fourier-transform infrared spectroscopic study of the polymorphic phase behavior of 1,2- bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine; a polymerizable lipid which forms novel microstructures

We have investigated the phase characteristics of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC23PC), a phosphatidylcholine with diacetylenic groups in the acyl chains, and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC), using Fourier-transform infrared spectroscopy (FTIR). Previous studies on the phase behavior of DC23PC in H2O have shown that DC23PC exhibits: (1) formation of cylindrical structures ('tubules') by cooling fluid phase multilamellar vesicles (MLVs) through Tm (43 degrees C), and 2) metastability of small unilamellar vesicles (SUVs) in the liquid-crystalline state some 40 degrees C below Tm, with subsequent formation of a gel phase comprised of multilamellar sheets at 2 degrees C. The sheets form tubules when heated and cooled through Tm. FTIR results presented here indicate that as metastable SUVs are cooled toward the transition to bilayer sheets, spectroscopic changes occur before the calorimetric transition as measured by a reduction in the CH2 symmetric stretch frequency and bandwidth. In spite of the vastly different morphologies, the sheet gel phase formed from SUVs is spectroscopically similar to the tubule gel phase. The C-H stretch region of DC23PC gel phase shows bands at 2937 and 2810 cm-1 not observed in the saturated analog of DC23PC, which may be related to perturbations in the acyl chains introduced by the diacetylenic moiety. The narrow CH2 scissoring mode at 1470 cm-1 and the prominent CH2 wagging progression indicate that DC23PC gel phase was highly ordered acyl chains with extended regions of all-trans methylene segments. In addition, the 13 cm-1 reduction in the C = O stretch frequency (1733-1720 cm-1) during the induction of DC23PC gel phase indicates that the interfacial region is dehydrated and rigid in the gel phase.     

Comparison of the effects of NaCl on the thermotropic behaviour of sn-1' and sn-3' stereoisomers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol

The phase behaviour of liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG) and the corresponding sn-3' stereoisomer (3'-DMPG) were studied by DSC as a function of NaCl concentration. The melting of the metastable gel phase to the liquid-crystalline phase was similar for both lipids. However, in the presence of salt and at 6 degrees C (T less than Tp) the gel phase of both stereoisomers of DMPG was shown to be metastable and a new phase nominated here as the highly crystalline phase was formed as the stable state. However, significant differences in the formation and melting of the highly crystalline phase were evident between the two polar headgroup stereoisomers. For 3'-DMPG in the presence of 300 mM NaCl the melting enthalpy of this phase is approx. 82 kJ/mol and the transition temperature about 11 degrees higher (at 33.6 degrees C) than for the gel to liquid-crystalline phase transition (25 kJ/mol at 23.0 degrees C). In the presence of 0.15-1.2 M NaCl at 6 to 10 degrees C the formation of the highly crystalline phase of 3'-DMPG is complete within 2 to 5 days, increasing [NaCl] facilitates the rate. For a 1:1 mixture of 1'- and 3'-DMPG the formation of the highly crystalline phase requires several weeks and melts at about 20 degrees higher than the gel phase (at approx. 40 degrees C). For 1'-DMPG partial conversion into the highly crystalline phase requires several months. For 3'-DMPG several intermediate phases appeared as endothermic peaks between the main phase transition temperature and the melting temperature of the highly crystalline phase. In contrast, for 1'-DMPG and the 1:1 mixture the subgel phase appears to be the only metastable intermediate phase. Different monovalent cations differ in their effect on the metastable behaviour.     

New structural model for mixed-chain phosphatidylcholine bilayers

Multilamellar suspensions of a mixed-chain saturated phosphatidylcholine with 18 carbon atoms in the sn-1 chain and 10 carbon atoms in the sn-2 chain have been analyzed by X-ray diffraction techniques. The structural parameters for this lipid in the gel state are quite different than usual phosphatidylcholine bilayer phases. A symmetric and sharp wide-angle reflection at 4.11 A indicates that the hydrocarbon chains in hydrated C(18):C(10)PC bilayers are more tightly packed than in usual gel-state phosphatidylcholine bilayers and that there is no hydrocarbon chain tilt. The lipid thickness is about 12 A smaller than would be expected in a normal bilayer phase, and the area per molecule is 3 times the area per hydrocarbon chain. In addition, the bilayer thickness increases upon melting to the liquid-crystalline state, whereas normal bilayer phases decrease in thickness upon melting. On the basis of these data, we propose a new lipid packing model for gel-state C(18):C(10)PC bilayers in which the long C(18) chain spans the entire width of the hydrocarbon region of the bilayer and the short C(10) chain aligns or abuts with the C(10) chain from the apposing molecule. This model is novel in that there are three hydrocarbon chains per head group at the lipid-water interface. Calculations show that this phase is energetically favorable for mixed-chain lipids provided the long acyl chain is nearly twice the length of the shorter chain. In the liquid-crystalline state C(18):C(10)PC forms a normal fluid bilayer, with two chains per head group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid

A comparative study of the polymorphism exhibited by the polymerizable, tubule-forming phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3- phosphocholine (DC23PC) and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC) in aqueous suspension is reported. Differential scanning calorimetry (DSC), as well as freeze-fracture electron microscopy and Raman spectroscopy, have been used to study the influence on phase behavior of rigid diacetylene groups in the fatty acyl chains of a phosphatidylcholine. DTPC large multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) suspensions were found to retain liposome morphology after chain crystallization had occurred. In marked contrast, diacetylenic DC23PC suspensions do not maintain liposomal morphology in converting to the low temperature phase. Large MLVs of DC23PC with outer diameters in excess of 1 micron convert to a gel phase with cylindrical or tubular morphology at 38 degrees C, just a few degrees below the lipid's chain melting temperature (TM(H), i.e. temperature of an endothermic event observed during a heating scan) of 43.1 degrees C. Unlike the large MLVs, small MLVs or SUVs of DC23PC, with diameters of 0.4 +/- 0.3 micron and 0.04 +/- 0.02 micron, respectively, exhibit metastability in the liquid-crystalline state for several tens of degrees below the chain melting temperature prior to converting to a gel phase which, by electron microscopy, manifests itself as extended multilamellar sheets. Raman data collected at TM(H) -40 degrees C demonstrate that the gel state formed by DC23PC is very highly ordered relative to that of DTPC, suggesting that special chain packing requirements are responsible for the novel phase behavior of DC23PC.     

Metastability and polymorphism in the gel phase of 1,2-dipalmitoyl-3-SN-phosphatidylcholine. A Fourier transform infrared study of the subtransition.

Fourier transform infrared spectroscopy was used to study the metastability of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) at temperatures near 0 degrees C. It was found that when DPPC is incubated at 2 degrees C for three days the two-dimensional acyl chain packing changes from one resulting in spectra typical of an orthorhombic subcell to one resembling that found in triclinically packed acyl systems. This transition proceeds in two stages. The first step, requiring less than one day, approximates first-order kinetics; the second stage proceeds with second- or higher-order kinetics. Comparison of spectra recorded at -36 degrees C with and without prior incubation at 2 degrees C shows that there are two stable low temperature forms of DPPC; that is, DPPC is metastable only within a narrow temperature range. A study of the thermotropic behavior in the range 0-45 degrees C shows that the subtransition near 15 degrees C is a transition from the alternate form to one with orthorhombic characteristics. Spectral changes at the pretransition and the main phase transition demonstrate that there are differences in behavior that are related to the thermal history of the sample.     

Phase behavior of galactocerebrosides from bovine brain

Bovine brain cerebrosides (BOV-CER) were separated by high-performance liquid chromatography into cerebroside fractions with a single acyl chain type or with a relatively homogeneous acyl chain distribution. The thermal behavior of these isolated cerebroside fractions was studied by differential scanning calorimetry. Nonhydroxy (n-acyl) fatty acid cerebrosides (NFA-CER) possessing a saturated acyl chain (C16:0, C18:0, C24:0) exhibit their major order-disorder transition temperature TM at 83 degrees C, independent of chain length. NFA-CER possessing primarily unsaturated acyl chains (C24:1) exhibits TM at 70 degrees C. 2-Hydroxy fatty acid cerebrosides (HFA-CER), which possess a saturated hydroxyacyl chain (C18:0h, C24:0h), exhibit TM at 70-72 degrees C. Thus, naturally occurring cerebrosides exhibit high TM's that do not depend significantly on acyl chain length and that depend only to a small degree on unsaturation and the presence of a 2-hydroxy branch in the amide-linked chain. Isolated NFA-CER's each exhibit metastable polymorphism of the type previously described for unfractionated NFA-CER [Curatolo, W. (1982) Biochemistry 21, 1761]. Polymorphism in HFA-CER is complex, with a different type of thermal behavior observed for each isolated acyl chain fraction studied. On prolonged storage at low temperature, unfractionated HFA-CER and unfractionated BOV-CER reach a highly ordered gel state similar to that which is readily reached by NFA-CER's. These results indicate that all cerebrosides exhibit metastable polymorphism. However, the kinetic barriers to reaching the stable gel state are greater for HFA-CER and BOV-CER than for NFA-CER.     

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)&z. Gt;Mg(2+)>Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.     

Conformation of phosphatidylcholine in neat and cholesterol-containing liquid-crystalline bilayers. Application of a novel method.

The conformation of phosphatidylcholine in liquid-crystalline bilayers was studied with a novel, high-resolution method employing phosphatidylcholine species containing pyrenyl moieties in both acyl chains of variable length. Analysis of the intramolecular pyrene-pyrene collision data obtained for 30 such species in terms of a simple geometrical model showed that the sn-1 acyl chain penetrates, on the average, 0.84 +/- 0.11 methylene units (0.8 A) deeper into the bilayer than the sn-2 chain at 22 degrees C. A similar value was obtained at 37 degrees C. Since the penetration difference of the sn-1 and sn-2 acyl chains is inherently coupled to the conformation of the glycerol moiety, these data mean that the glycerol moiety of phosphatidylcholine is, on the average, only moderately tilted with respect to the bilayer plane in the liquid-crystalline state. This contrasts the perpendicular orientation observed previously for phosphatidylcholine crystals [Pearson, R. H., & Pascher, I. (1979) Nature 281, 499-501]. Importantly, addition of 50 mol % cholesterol, which is known to reduce dramatically the interactions between phosphatidylcholine molecules in bilayers, had only a small effect on the penetration difference of the acyl chains, strongly suggesting that the conformation of phosphatidylcholine in the liquid-crystalline state is determined largely by intramolecular, rather than intermolecular, interactions.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

Incorporation and remodeling of phosphatidylethanolamine containing short acyl residues in yeast

Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn− 2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn− 2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.     

Structural transitions in short-chain lipid assemblies studied by (31)P-NMR spectroscopy

The self-assembled supramolecular structures of diacylphosphatidylcholine (diC(n)PC), diacylphosphatidylethanolamine (diC(n)PE), diacylphosphatidyglycerol (diC(n)PG), and diacylphosphatidylserine (diC(n)PS) were investigated by (31)P nuclear magnetic resonance (NMR) spectroscopy as a function of the hydrophobic acyl chain length. Short-chain homologs of these lipids formed micelles, and longer-chain homologs formed bilayers. The shortest acyl chain lengths that supported bilayer structures depended on the headgroup of the lipids. They increased in the order PE (C(6)) < PC (C(9)) < or = PS (C(9) or C(10)) < PG (C(11) or C(12)). This order correlated with the effective headgroup area, which is a function of the physical size, charge, hydration, and hydrogen-bonding capacity of the four headgroups. Electrostatic screening of the headgroup charge with NaCl reduced the effective headgroup area of PS and PG and thereby decreased the micelle-to-bilayer transition of these lipid classes to shorter chain lengths. The experimentally determined supramolecular structures were compared to the assembly states predicted by packing constraints that were calculated from the hydrocarbon-chain volume and effective headgroup area of each lipid. The model accurately predicted the chain-length threshold for bilayer formation if the relative displacement of the acyl chains of the phospholipid were taken into account. The model also predicted cylindrical rather than spherical micelles for all four diacylphospholipid classes and the (31)P-NMR spectra provided evidence for a tubular network that appeared as an intermediate phase at the micelle-to-bilayer transition. The free energy of micellization per methylene group was independent of the structure of the supramolecular assembly, but was -0.95 kJ/mol (-0.23 kcal/mol) for the PGs compared to -2.5 kJ/mol (-0.60 kcal/mol) for the PCs. The integral membrane protein OmpA did not change the bilayer structure of thin (diC(10)PC) bilayers.     

Binary mixtures of saturated and unsaturated mixed-chain phosphatidylcholine. A differential scanning calorimetry study

High-resolution differential scanning calorimetry (DSC) has been used to study the aqueous dispersions of mixed-chain phosphatidylcholines prepared from colyophilized mixtures of C(18):C(11:1 delta 10) PC/C(18):C(10)PC and C(18):C(11:1 delta 10) PC/C(18):C(11)PC of various molar ratios. These mixed-chain phospholipids are characterized by a marked disparity in their acyl-chain lengths; however, the sn-1 acyl chain in the fully extended conformation is about twice as long as the sn-2 acyl chain. Their thermotropic behavior was determined, and the phase diagrams of these two mixtures were constructed from the calorimetric data. Results indicate that C(18):C(11:1 delta 10)PC/C(18):C(10)PC and C(18):C-(11:1 delta 10)PC/C(18):C(11)PC are miscible in all proportions with a near-ideal behavior of mixing in the gel and liquid-crystalline phases. Equimolar mixtures of diC(14)PC/C(18):C(11:1 delta 10)PC, diC(14)PC/C(18):C(10)PC, and diC(14)PC/C(18):C(11)PC have also been studied by DSC. These phosphatidylcholines in the 1:1 mixture differ in Tm by less than 11 degrees C; however, they exhibit gel-phase immiscibility in the plane of the bilayer. Taken together, these studies suggest that C(18):C(11)PC and C(18):C(11:1 delta 10)PC are packed similarly to C(18):C(10)PC in excess water as mixed interdigitated bilayers, at T less than Tm, which transform into partially interdigitated bilayers when heated above Tm.     

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.     

Calorimetric studies on the influence of N-methylated headgroups on the mixing behavior of diheptadecanoyl phosphatidylcholine with 1-behenoyl-2-lauroylphosphatidylcholine.

Recent studies of five different phosphatidylcholine/phosphatidylcholine (PC/PC) systems indicate that binary mixtures of phosphatidylcholines in which one component has a normalized chain length difference (delta C/CL) in the range of 0.09-0.40 and the other a delta C/CL in the range of 0.42-0.57 exhibit the phase behavior of a eutectic system. Here, delta C is the effective chain-length difference between the two acyl chains, and CL is the effective length of the longer of the two acyl chains for the same lipid molecule in the gel state. In each mixture, gel phase immiscibility occurs over a wide compositional range due to the difference in the gel phase acyl chain packing properties of each component. Although the mixtures differ in the location of their eutectic horizontal, with respect to temperature, all have a similar eutectic point that occurs at a composition of approximately 40 mol percent of the component with the delta C/CL value in the range of 0.42-0.57. Here, we extend these studies by systematically modifying the headgroup of C(17):C(17)PC and then analyzing the mixing behavior of the modified lipid with C(22):C(12)PC using DSC. Progressive demethylation of the C(17):C(17)PC headgroup leads to an increase in gel phase immiscibility and a decrease in the amount of C(22):C(12)PC that comprises the eutectic composition. The temperature defining the location of the eutectic horizontal, however, remains virtually unchanged in all three phase diagrams. Our results suggest that the eutectic composition is influenced by changes in gel phase acyl chain packing that are dependent on headgroup-headgroup interactions. In contrast, the eutectic nature of the phase diagram and the location of its solidus line are properties of acyl chain interactions that are independent of phospholipid headgroup-headgroup interactions.     

Study of the effect of melittin on the thermotropism of dipalmitoylphosphatidylcholine by Raman spectroscopy

The effect of amphiphilic toxin melittin (Mel) on the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) has been studied by Raman spectroscopy. The spectra show that for complexes that were incubated above 40 degrees C, melittin does not penetrate DPPC bilayers in the gel state as an intrinsic protein since the conformation of the lipid acyl chains is just slightly perturbed by the toxin. Instead, at the DPPC/Mel molar ratios investigated (Ri = 5 and 15), Raman results suggest the formation of discoidal particles as complexes of apolipoproteins with phosphatidylcholines. These lipid/protein assemblies are characterized by a high conformational order, low intermolecular chain-chain interactions due to the size of the particles, and a low cooperativity of the gel to liquid-crystalline transition. The latter is biphasic for samples studied. It is believed that aggregation of these particles into larger ones occurs when the bilayers become less stable at higher temperature and that melittin is partially embedded into the hydrophobic core of the larger lipid/protein units. The freezing of the dispersion at approximately 0 degrees C also causes a reversible aggregation of the particles that leads to the formation of domains in which the interchain interactions are very similar to that of the pure lipid. The small particles of DPPC/Mel are also metastable, and with time, they form larger aggregates from which melittin is expulsed.