The mixing behavior of pseudobinary phosphatidylcholine-phosphatidylglycerol mixtures as a function of pH and chain length

The miscibility of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with different chain lengths (n = 14, 16) was examined by differential scanning calorimetry (DSC) at pH 2 and pH 7. The determination of the coexistence curves of the phase diagrams was performed using a new procedure, namely the direct simulation of the heat capacity curves as described recently (Johann et al. 1996, Garidel et al. 1997). From the simulations of the heat capacity curves first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained and phase diagrams were constructed using temperatures for the onset and offset of melting which were corrected for the broadening effect caused by a decrease in cooperativity of the transition. In most cases, the composition dependence of the nonideality parameters indicated nonsymmetric mixing behavior. The phase diagrams were further refined by simulations of the coexisting curves using a four-parameter model to account for nonideal and nonsymmetric mixing in the gel as well as in the liquid-crystalline phase. The mixing behavior of the systems was analyzed as a function of pH and chain length difference to elucidate the effect of these two parameters on the shape of the phase diagrams. At pH 7 the phase boundaries are much closer together and a narrower coexistence range is obtained compared to the corresponding phase diagrams at pH 2. For DPPC/DMPG at pH 2, the shape of the phase diagram and the strongly positive nonideality parameter ρ ₁ for the liquid-crystalline phase indicates an upper azeotropic point. This indicates an unusual behavior of the system, namely more pronounced clustering of like molecules in the liquid-crystalline phase compared to the gel phase. Received: 17 March 1997 / Accepted: 4 July 1997     

New approaches to the simulation of heat-capacity curves and phase diagrams of pseudobinary phospholipid mixtures.

A simulation program using least-squares minimization was developed to calculate and fit heat capacity (cp) curves to experimental thermograms of dilute aqueous dispersions of phospholipid mixtures determined by high-sensitivity differential scanning calorimetry. We analyzed cp curves and phase diagrams of the pseudobinary aqueous lipid systems 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol/ 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DMPG/DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid/1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DMPA/DPPC) at pH 7. The simulation of the cp curves is based on regular solution theory using two nonideality parameters rho g and rho l for symmetric nonideal mixing in the gel and the liquid-crystalline phases. The broadening of the cp curves owing to limited cooperativity is incorporated into the simulation by convolution of the cp curves calculated for infinite cooperativity with a broadening function derived from a simple two-state transition model with the cooperative unit size n = delta HVH/delta Hcal as an adjustable parameter. The nonideality parameters and the cooperative unit size turn out to be functions of composition. In a second step, phase diagrams were calculated and fitted to the experimental data by use of regular solution theory with four different model assumptions. The best fits were obtained with a four-parameter model based on nonsymmetric, nonideal mixing in both phases. The simulations of the phase diagrams show that the absolute values of the nonideality parameters can be changed in a certain range without large effects on the shape of the phase diagram as long as the difference of the nonideality parameters for rho g for the gel and rho l for the liquid-crystalline phase remains constant. The miscibility in DMPG/DPPC and DMPA/DPPC mixtures differs remarkably because, for DMPG/DPPC, delta rho = rho l -rho g is negative, whereas for DMPA/DPPC this difference is positive. For DMPA/DPPC, this difference is interpreted as being caused by a negative rho g value, indicating complex formation of unlike molecules in the gel phase.     

Non-ideal mixing and fluid–fluid immiscibility in phosphatidic acid–phosphatidylethanolamine mixed bilayers

Differential scanning calorimetry (DSC) was used to study the miscibility of phosphatidic acids (PAs) with phosphatidylethanolamines (PEs) as a function of chain length (n = 14, 16) and degree of ionization of PAs at pH 4, pH 7, and pH 12. Phase diagrams were constructed using temperature data for onset and end of the phase transition obtained from the direct simulation of the heat-capacity curves. The phase diagrams were analyzed by simulations of the coexistence curves utilizing a four-parameter regular solution model. For PA–PE mixtures, the non-ideality parameters are a function of composition indicating non-symmetric non-ideal mixing behavior. At pH 7, where the PA component is negatively charged, the systems DMPA:DMPE and DPPA:DPPE have positive non-ideality parameters ρ ₁ in both phases, indicating a preferred aggregation of like molecules. In contrast, DMPA:DPPE and DPPA:DMPE mixtures had negative ρ ₁ values. Measurements at pH 4 showed that mixed pair formation is favored when PA is protonated. At pH 12 where PA is doubly charged, highly positive ρ _l1 parameters are obtained for the liquid-crystalline phase except for the system DPPA:DPPE (ρ ₁ < 0). This indicates clustering of like molecules and possibly domain formation in the liquid-crystalline phase. DPPA:DMPE at pH 12 even shows a miscibility gap in the liquid-crystalline phase. Obviously, despite the presence of doubly charged PA a fluid–fluid immiscibility is induced.     

Packing characteristics of a model system mimicking cytoplasmic bacterial membranes.

The phase diagram of fully hydrated mixtures of dipalmitoylphosphatidylethanolamine and -phosphatidylglycerol was constructed and the coexistence lines of the solidus and liquidus curve calculated based on regular solution theory using two nonideality parameters for each of the phase to account for nonideal and nonsymmetric mixing. Both lipids show nonideal miscibility in the liquid-crystalline phase, while a region of immiscibility exists in the lamellar-gel phase between the mole fraction x(DPPE)=0.05-0.4. Two lines of three-phase coexistence around 35 and 40 degrees C reflects the presence of lipid domains predominantly composed of phosphatidylglycerol as well as of the mixed lipid system. This is reflected in the positive nonideality parameters of the gel phase obtained from the simulation of the phase diagram. Moreover, segregation of pure phosphatidylethanolamine domains was detected in mixtures x(DPPE)>0.9, which formed multilamellar liposomes, while unilamellarity was observed for the mixed lipid systems owing to the presence of the negatively charged phosphatidylglycerol. The packing constraints of these phospholipids, major components of cytoplasmic bacterial membranes, may be of importance in the interaction with various solutes like antimicrobial peptides, and were explained based on the nature of the headgroups and the molecular geometry of the phospholipids.     

Calcium-induced lipid phase separations and interactions of phosphatidylcholine/anionic phospholipid vesicles. Fluorescence studies using carbazole-labeled and brominated phospholipids

A novel method that uses a carbazole-labeled fluorescent phosphatidylcholine, which partitions preferentially into liquid-crystalline lipid domains, to monitor the kinetics and the extents of thermotropic and ionotropic lateral phase separations in vesicles combining brominated and nonbrominated phosphatidylcholines (PCs), phosphatidic acids (PAs), and phosphatidylserines (PSs) is described. The calcium-induced segregation of several nonbrominated PA species in liquid-crystalline brominated PC bilayers behaves as a well-defined lateral phase separation; the residual solubility of the PA component in the PC-rich phase in the presence of calcium can vary severalfold depending on the PA acyl chain composition. PC/PS mixtures show a pronounced tendency to form metastable solutions in the presence of calcium, particularly when they contain less than equimolar proportions of PS. This metastability is not readily relaxed by repeated freeze-thawing of vesicles in the presence of calcium, by avidin-mediated contacts between PC/PS vesicles containing biotinylated lipids, or by calcium-induced lateral segregation of PA in the same vesicles. Different PS species exhibit different apparent residual solubilities in liquid-crystalline PC bilayers, ranging from less than 10 mol % for dimyristoyl-PS to ca. 45 mol% for dioleoyl-PS, after prolonged incubations of PC/PS multilamellar vesicles with excess calcium. Results are presented, obtained by using the above lipid-segregation assay and parallel assays of intervesicle lipid mixing, that raise questions concerning the relevance of the equilibrium behavior of calcium-treated PS/PC mixtures to the relatively rapid interactions (fusion and lipid mixing) of PC/PS vesicles that follow initial exposure to calcium.     

Miscibility of phosphatidylethanolamine-phosphatidylglycerol mixtures as a function of pH and acyl chain length

We have examined the mixing properties of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the major components of many bacterial membranes. The phase transition behavior of dilute aqueous suspensions of PE:PG mixtures with different chain lengths (n = 14, 16) in 0.1 M NaCl at pH 7 and pH 2 was investigated by differential scanning calorimetry (DSC). The DSC curves were simulated using an approach which takes into account the broadening of the phase transition in addition to symmetric, non-ideal mixing in the gel and the liquid-crystalline phase. Based on the temperatures for onset and end of “melting” obtained by the simulations, the phase diagrams were constructed and then refined using a regular solution model with non-symmetric mixing in both phases. The mixing properties of PE:PG mixtures were analyzed as a function of pH and acyl chain length. In almost all cases, non-symmetric mixing behavior was observed, i.e. the non-ideality parameters are different for bilayers with low PG content compared to bilayers with high PG content. For equimolar mixtures at pH 7, when PG is negatively charged, the non-ideality parameters are negative for both phases, indicating preferential formation of mixed pairs. This mixed pair formation is more pronounced for the gel phase. At pH 2, when PG is partly protonated, the non-ideality parameter is less negative and the formation of mixed pairs is reduced compared to pH 7. The formation of PE:PG mixed pairs at pH 7 might be of benefit to a bacterial membrane, because it prevents demixing of lipid components with a concomitant destabilization of the membrane. Received: 3 August 1998 / Revised version: 4 October 1999 / Accepted: 12 October 1999     

The effect of nonideal lateral mixing on the transmembrane lipid asymmetry

It is shown that the equilibrium transmembrane lipid asymmetry strongly depends on the degree of nonideality in the lateral mixing of the lipid components. In two-component bilayers the effect of nonideal lateral mixing is maximal for a given component at mole fractions of this component between 0.35 and 0.4. For asymmetry creating factors about 3 kT correcting for lateral nonidealities typical for lipids can increase as much as three times the transmembrane asymmetry. The relationship between lateral nonideality and transbilayer asymmetry is analysed in detail in the case of electrostatically induced asymmetry by using the Gouy-Chapman theory of electric double layers and the Bragg-Williams (regular solutions) approximation of nonideal lateral mixing. Two representative models are studied: (a) a single flat bilayer with a transmembrane electric potential difference applied on it; (b) two parallel membranes at short separation. In case (a), for transmembrane potentials of about 50-100 mV the introduction of nonideality corrections increases up to 40% the transmembrane asymmetry. In case (b), at physiological electrolyte concentrations the lipid asymmetry and, consequently, the effect of lateral nonideality become significant only at unrealistically small separations between the membranes. The surprisingly great influence of the lateral nonideality on the equilibrium transmembrane asymmetry suggests a significant role for this effect in determining the membrane molecular organization. A restricted lateral lipid miscibility might serve as a peculiar, but rather strong 'amplifier' of the transmembrane asymmetry. The qualitatively different asymmetries found in small unilamellar phosphatidylcholine-phosphatidylethanolamine vesicles of different fatty acid composition (Lentz, B.R. and Litman, B.J. (1978) Biochemistry 17, 5537-5543) can be reasonably well explained as an effect of the lateral nonideality. A hypothesis considering the transmembrane distributions of the major phospholipid species in erythrocytes as evolving from their lateral miscibilities is proposed.     

Non-linear least squares analysis of phase diagrams for non-ideal binary mixtures of phospholipids

A computer program for non-linear least squares minimization has been applied to construct temperature-composition phase diagrams for several binary systems of different phospholipids based on their calorimetric data. The calculated phase diagram is guided to fit the calorimetric data with two adjustable parameters that describe the non-ideal mixing of lipid components in the gel and liquid-crystalline phases. The parameter estimation procedure is presented to show that the computer program can be used not only to generate phase diagrams with characteristic shapes but also to numerically estimate the lipid-lipid pair interactions between the mixed and the like pairs in the two-dimensional plane of the bilayer in both the gel and liquid-crystalline states. The binary lipid systems examined include dimyristoylphosphatidylcholine/1-palmitoyl-2-stearoylphosphatidylchol ine, 1-capryl-2-behenoylphosphatidylcholine/1-behenoyl-2-lauro ylphosphatidylcholine, and 1-stearoyl-2-caprylphosphatidylcholine/dimyristoylphosphatidylchol ine.     

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)     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and 31P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (approximately 11-15 degrees C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (approximately 23-25 degrees C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing approximately 30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the Lc phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the Lc phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the Lc phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Kinetics and thermodynamics of calcium-induced lateral phase separations in phosphatidic acid containing bilayers

The effects of calcium on the mixing of synthetic diacylphosphatidylcholines (PC's) and diacylphosphatidylethanolamines (PE's) with the corresponding phosphatidic acids (PA's) have been examined by high-sensitivity differential scanning calorimetry and by measurements of the fluorescence of labeled PA or PC species in PA-PC bilayers. Calorimetrically derived phase diagrams for dimyristoyl- and dielaidoyl-substituted PA-PC and PA-PE mixtures indicate that these species are readily miscible in the absence of calcium but phase-separate very extensively in the presence of high levels of calcium (30 mM). The limiting solubilities of PA (Ca2+) in liquid-crystalline PC or PE bilayers are less than or equal to 10 and approximately 5 mol %, respectively, while approximately 20 mol % of PC or PE can be introduced into the "cochleate" phase of PA (Ca2+) before a distinct PC-rich (or PE-rich) phase appears. The kinetics of calcium-induced lateral phase separations were examined for dioleoyl- and dielaidoyl-substituted PA-PC unilamellar vesicles labeled with fluorescent (C12-NBD-acyl) PA or PC, whose fluorescence becomes partially quenched upon phase separation. Our results indicate that, for the PA-PC system, lateral phase separation is very rapid (approximately less than 1 s) after calcium addition and develops partially (possibly in only one face of the bilayer) when calcium is present only on one side of the bilayer. Moreover, phase separations can develop at a rate faster than that of vesicle diffusion when calcium is added to dilute suspensions of vesicles, suggesting that interbilayer contacts are not essential to promote phase separations.     

Mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers: evidence for gel and liquid-crystalline phase immiscibility

The mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers has been investigated by high-sensitivity differential scanning calorimetry. Phase diagrams have been constructed for two-component bilayers composed of C(18)C(18)PC and either C(18)C(16)PC, C(18)C(14)PC, C(18)C(12)PC, or C(18)C(10)PC. It is found that C(18)C(18)PC-C(18)C(16)PC and C(18)C(18)PC-C(18)C(14)PC mixed bilayers exhibit complete miscibility of the components in both the gel and liquid-crystalline phases. Whereas this mixing is observed to be nearly ideal for the C(18)C(18)PC-C(18)C(16)PC binary system, the intermixing of the lipids is highly nonideal in the gel phase of the C(18)C(18)PC-C(18)C(14)PC binary mixture. The C(18)C(18)PC-C(18)C(12)PC and C(18)C(18)PC-C(18)C(10)PC mixed bilayers are characterized by partial immiscibility of the phosphatidylcholine components in the bilayer gel phase. Over a large compositional range, these bilayers appear to consist of phase-separated regions of interdigitated and noninterdigitated gel phases. In addition, the C(18)C(18)PC-C(18)C(10)PC two-component bilayer displays a limited region of liquid-liquid immiscibility in the liquid-crystalline bilayer phase. The phase separation of the mixed chain length phosphatidylcholines revealed in these mixed bilayers may represent a three-dimensional phase separation of the lipid components where the phosphatidylcholines are both laterally separated within the plane of the bilayer and conformationally coupled across the bilayer. Such phase-separated domains could have profound effects on membrane structure and function if they were to occur in biological membranes.     

Differential scanning calorimetric studies of the thermotropic phase behavior of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC-DPPC bilayers at any composition, and that SOPC-DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC-DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC-DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC-DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.     

Anomalous mixing of zwitterionic and anionic phospholipids with double-chain cationic amphiphiles in lipid bilayers.

High-sensitivity scanning calorimetry has been used to examine the thermotropic behavior of mixtures combining dipalmitoylphosphatidylcholine (DPPC), phosphatidylethanolamine (DPPE) and O-methylphosphatidic acid (DPPA-OMe) with the double-chain cationic amphiphiles N,N-dihexadecyl-N,N- dimethylammonium chloride (DHDAC), 1,2-dipalmitoyloxy-3-(trimethylammonio)propane (DPTAP) and the corresponding monomethylated tertiary amino compounds (DHMMA-H+ and DPDAP-H+). At physiological ionic strength, mixtures of these cationic amphiphiles with the anionic phospholipid DPPA-OMe can show gel-to-liquid-crystalline phase transitions at considerably higher temperatures than do either of the pure components. Surprisingly, binary mixtures of DPPC and these cationic amphiphiles also show strongly nonideal mixing, with phase diagrams exhibiting pronounced maxima in their solidus and liquidus curves. Similar behavior is not observed for mixtures of DPPC with DPPA-OMe, which closely resembles DPTAP and DPDAP-H+ in backbone configuration and polar headgroup size. The present results suggest that perturbation of the orientation of the phosphatidylcholine headgroup by cationic amphiphiles, as demonstrated previously by Seelig and co-workers (Biochemistry 28 [1989], 7720-7728), can significantly affect the thermotropic behavior of phospholipids such as DPPC. Such effects may exert a generally important (though not always easily recognizable) influence on the organization and thermotropic behavior of systems where zwitterionic phospholipids are combined with charged bilayer-associated molecules.     

Apparently anomalous sedimentation behavior in mixed solvent systems with strong interactions between solution components: analysis of nonideal behavior by bovine serum albumin in 7 M urea at pH 3.3

The use of the analytical ultracentrifuge to study nonideal behavior of macromolecules in multicomponent systems is discussed, noting the value of interference optics to extend the range of concentrations of macromolecule that may be studied. The choice of appropriate theory in the treatment of experimental data is examined, using a study of bovine serum albumin (BSA) in 7 M urea at pH 3.3 as an example. Under these conditions BSA undergoes extensive unfolding and exhibits marked nonideality, with the binding of approximately 200 molecules of urea per molecule of BSA.     

Lateral distribution of a pyrene-labeled phosphatidylcholine in phosphatidylcholine bilayers: fluorescence phase and modulation study

The lateral distribution of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PyrPC) was studied in small unilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions. The DPPC and DMPC experiments were carried out over temperature ranges above and below the matrix phospholipid phase transition temperature (Tm). The excimer to monomer fluorescence intensity ratio (E/M) was determined as a function of temperature for the three PyrPC/lipid mixtures. Phase and modulation data were used to determine the temperature dependence of pyrene fluorescence rate parameters in gel and in liquid-crystalline bilayers. These parameters were then used to provide information about excited-state fluorescence in phospholipid bilayers, calculate the concentration of the probe within liquid-crystalline and gel domains in the phase transition region of PyrPC in DPPC, and simulate E/M vs. temperature curves for three systems whose phase diagrams are different. From the simulated curves we could determine the relationship between the shape of the three simulated E/M vs. temperature curves and the lateral distribution of the probe. This information was then used to interpret the three experimentally derived E/M vs. temperature curves. Our results indicate that PyrPC is randomly distributed in pure gel and fluid phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and ³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11-15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23-25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Fourier-transform infrared studies of CaATPase partitioning in phospholipid mixtures of 1,2-dipalmitoylphosphatidylcholine-d62 with 1-palmitoyl-2-oleoylphosphatidylethanolamine and 1-stearoyl-2-oleoylphosphatidylcholine

CaATPase from rabbit sarcoplasmic reticulum has been reconstituted into binary lipid mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1,2-dipalmitoylphosphatidylcholine-d62 (DPPC-d62) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC)/DPPC-d62. Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor temperature-induced structural alterations in the individual lipid components in the presence and absence of protein. A simple two-state model is used to construct a phase diagram that is in good agreement with one constructed from differential scanning calorimetry data, for the POPE/DPPC-d62 (protein-free) system. Although these two lipids are miscible over at least most of the composition range, substantial deviations from ideal behavior are observed. An estimate of the nonideality of mixing in both the gel and liquid-crystalline phases is obtained from regular solution theory. The phase diagram for SOPC/DPPC-d62 shows gel-phase immiscibility. FT-IR studies of ternary (POPE/DPPC-d62/CaATPase) complexes indicate that both lipid components are disordered by protein at all temperatures studied. In addition, their melting events are broadened and shifted to lower temperatures compared with the appropriate binary lipid mixture. Semiquantitative estimates for the fraction of each lipid melted are obtained from the model. The effect of protein on SOPC/DPPC-d62 mixtures depends on that total lipid to protein ratio. At low protein levels, SOPC is preferentially selected by CaATPase, so that bulk lipid is enriched in DPPC-d62. At high levels of protein, both lipid components are selected. The applicability of vibrational spectroscopy for determination of the partitioning preferences of membrane proteins into regions of particular chemical structure or physical order in a complex lipid environment is demonstrated.