SANS study on the effect of lanthanide ions and charged lipids on the morphology of phospholipid mixtures. Small-angle neutron scattering

The structural phase behavior of phospholipid mixtures consisting of short-chain (dihexanoyl phosphatidylcholine) and long-chain lipids (dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol), with and without lanthanide ions was investigated by small-angle neutron scattering (SANS). SANS profiles were obtained from 10 degrees C to 55 degrees C using lipid concentrations ranging from 0.0025 g/ml to 0.25 g/ml. The results reveal a wealth of distinct morphologies, including lamellae, multi-lamellar vesicles, unilamellar vesicles, and bicellar disks.     

The peculiar thermo-structural behavior of the anionic lipid DMPG

Aqueous dispersions of the anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG), around 100 mM ionic strength, are known to exhibit a thermal behavior similar to that of the largely studied lipid dimyristoyl phosphatidylcholine (DMPC), which undergoes a gel to liquid crystalline phase transition at 23 degrees C, well characterized by differential scanning calorimetry (DSC), and other methods. However, at low ionic strength, DMPG has been shown to present a large gel-fluid transition region, ranging from 18 to 35 degrees C. This intermediate phase is optically transparent and characterized by a continuous change in membrane packing. Structural properties of the DMPG gel-fluid transition region will be discussed, based on results obtained by several techniques: electron spin resonance (ESR) of spin labels at the membrane surface and intercalated at different depths in the bilayer; light scattering; DSC; small angle X-ray scattering (SAXS); and fluorescence spectroscopy of probes in the bilayer.     

Effect of Lipid Head Groups on Double-Layered Two-Dimensional Crystals Formed by Aquaporin-0

Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.     

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [(14)C]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [(14)C]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains ((14)C atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosity of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel-fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases.     

Phase transition of dimyristoylphosphatidylglycerol bilayers induced by electric field pulses

The phase transition of dimyristoylphosphatidylglycerol (DMPG) bilayers has been studied by measurements of light scattering under high electric field pulses. Midpoints of phase transitions have been identified by a clear discontinuity of field induced relaxation amplitudes. We show that the phase transition of DMPG suspensions in monovalent salt is virtually independent of the electric field strength up to approx. 35 kV/cm. A shift of the lipid phase by electric field pulses has been observed, however, for DMPG suspensions in the presence of Ca2+ ions. DMPG suspensions exhibit a jump of the phase transition temperature from 17 degrees C at Ca/DMPG molar ratios r less than 1/7 to 32 degrees C at r greater than 1/7. Field pulses of 60 to 100 microseconds applied to DMPG suspensions with Ca2+ at r greater than 1/7 induce discontinuities of relaxation amplitudes in the temperature range 15 to 22 degrees C in addition to the 'standard' one at 32 degrees C, when the electric field strength is above 15 kV/cm. These results indicate that electric field pulses induce a transition from the phase formed at 'high' Ca(2+)- to the one formed at 'low' Ca(2+)-ion concentrations. Our results are consistent with a dissociation field effect on Ca(2+)-lipid complexes which drives the phase transition.     

Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.     

Thermal and structural behavior of dioctadecyldimethylammonium bromide dispersions studied by differential scanning calorimetry and x-ray scattering

Dioctadecyldimethylammonium bromide (DODAB) is a double chain cationic lipid, which assembles as bilayer structures in aqueous solution. The precise structures formed depend on, e.g., lipid concentration and temperature. We here combine differential scanning calorimetry (DSC) and X-ray scattering (SAXS and WAXS) to investigate the thermal and structural behavior of up to 120 mM DODAB in water within the temperature range 1–70°C. Below 1 mM, this system is dominated by unilamellar vesicles (ULVs). Between 1 and 65 mM, ULVs and multilamellar structures (MLSs) co-exist, while above 65 mM, the MLSs are the preferred structure. Depending on temperature, DSC and X-ray data show that the vesicles can be either in the subgel (SG), gel, or liquid crystalline (LC) state, while the MLSs (with lattice distance d  = 36.7 Å) consist of interdigitated lamellae in the SG state, and ULVs in the LC state (no Bragg peak). Critical temperatures related to the thermal transitions of these bilayer structures obtained in the heating and cooling modes are reported, together with the corresponding transition enthalpies.     

1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) monolayers: influence of temperature, pH, ionic strength and binding of alkaline earth cations

Ion binding and lipid ionization of the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in monolayers was studied by measuring the lateral pressure Pi as a function of the molecular area A at the air/water interface at different temperatures. The pH of the subphase (pH 2 and 7) and the ionic strength (NaCl) was varied. In addition, different divalent cations (1mM MgCl2, CaCl2 and SrCl2, pH 7) were added. DMPG is partly protonated on pure water at pH 7. An increase in the NaCl concentration in the subphase leads to film expansion. This effect is caused by an ionization of the headgroup of DMPG, i.e. a shift of the apparent pK. More condensed films are obtained on pure water at pH 2, due to the reduction of electrostatic repulsion by headgroup protonation and the possibility for the formation of a hydrogen bonding network. The divalent cations Mg2+, Ca2+ and Sr2+ interact differently with a DMPG monolayer in pure water at pH 7. In the presence of 1mM CaCl2 a condensation of the DMPG film is induced, whereas an expansion of the monolayer is observed in the presence of Mg2+ and Sr2+. Two counteracting effects are operative: (a) ionization of the headgroup due to electrostatic screening leads to film expansion and (b) binding of the divalent cations to the lipid headgroups leads to condensation. The latter effect is more pronounced in the case of Ca2+, whereas the binding of Mg2+ and Sr2+ to DMPG is weaker. Site-specific cation binding has to be assumed in addition to electrostatic effects.     

Influence of Ca2+ and Mg2+ on the thermotropic behaviour and permeability properties of liposomes prepared from dimyristoyl phosphatidylglycerol and mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine.

Calorimetric experiments showed a marked effect of Ca2+ and Mg2+ on the thermotropic behaviour of dimyristoyl phosphatidylglycerol. 2. Concentrations of Ca2+ and Mg2+ lower than 1 ion to 2 molecules of phosphatidylglycerol produced a shift of the phase transition to higher temperatures and an increase in the enthalpy change which is consistent with a closer packing of the lipid molecules in the liposomes. 3. Above the 1:2 ratio, freeze-fracture electron microscopy demonstrated typical "crystal" structures both in the presence of Ca2+ and Mg2+. In the presence of Mg2+ a metastable behaviour was noticed in the calorimetric experiments. 4. A Ca2+- and Mg2+-induced shift in the transition temperature and an increase in the enthalpy change was also observed in a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine. However, these mixed samples remained liposomal in structure at any concentration of the divalent ions. 5. Liposomes prepared from a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the absence of divalent cations are permeable in the range 10-50 degrees C. Bilayers of mixtures neutralized by Ca2+ or Mg2+ were demonstrated to be completely impermeable to K+, except in the vicinity of the phase transition. 6. The leak of ions from liposomes of a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the vicinity of the phase transition temperature was considerably less in the presence of Ca2+ than in the presence of Mg2+. 7. It is concluded that there is a correlation between the calorimetric data and the permeability properties of dimyristoyl phosphatidylglycerol-containing bilayers with respect to the influence of Ca2+ and Mg2+.     

The effects of temperature,salinity, concentration and PEGylated lipid on the spontaneous nanostructures of bicellar mixtures

The self-assembling morphologies of low-concentration (mostly 1 and 10 mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C₁₆) phosphatidylcholine (DPPC), dihexanoyl (di-C₆) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C₁₆) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mol% of the total lipid composition. The effects of several parameters on the spontaneous structures were studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, T_M of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition was found upon the elevation of temperature (above T_M) in the cases of low lipid concentration in the absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE, and the amount of DHPC located at the disc rim.     

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

Does sucrose influence the properties of DMPC vesicles?

Small-angle neutron and X-ray scattering, dynamic light scattering, X-ray diffraction coupled with differential scanning calorimetry, and Raman spectroscopy were applied to investigate unilamellar (ULVs) and multilamellar (MLVs) dimyristoylphosphatidylcholine (DMPC) vesicles in aqueous sucrose solutions with sucrose concentrations from 0 to 60% w/w. In case of ULVs, the addition of sucrose decreases the polydispersity of vesicle population. A minimum value of polydispersity was found at 20% sucrose. For sucrose concentration from 0 to 35% oligolamellar vesicles in the ULV population have a minimum presence. Vesicles with 5-10% sucrose exhibit the best stability in time. For the case of MLVs, sucrose influences the temperature of the phase transitions, but the internal membrane structure remains unchanged.     

Acidic phospholipid bicelles: a versatile model membrane system

With the aim of establishing acidic bicellar solutions as a useful membrane model system, we have used deuterium NMR spectroscopy to investigate the properties of dimyristoyl/dihexanoylphosphatidylcholine (DMPC/DHPC) bicelles containing 25% (w/w in H(2)O) of either dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG). The addition of the acidic lipid component to this lyotropic liquid crystalline system reduces its range of stability because of poor miscibility of the two dimyristoylated phospholipids. Compared to the neutral bicelles, which are stable at pH 4 to pH 7, acidic bicelles are stable only from pH 5.5 to pH 7. Solid-state deuterium NMR analysis of d(54)-DMPC showed similar ordering in neutral and acidic bicelles. Fully deuterated DMPS or DMPG is ordered in a way similar to that of DMPC. Study of the binding of the myristoylated N-terminal 14-residue peptide mu-GSSKSKPKDPSQRR from pp60(nu-src) to both neutral and acidic bicelles shows the utility of these novel membrane mimetics.     

Direct measurement of the interaction between phosphatidylglycerol bilayers in aqueous electrolyte solutions.

Results are presented of force measurements between deposited bilayers of dimyristoylphosphatidyl glycerol (DMPG) at T greater than Tm, and distearoylphosphatidyl glycerol (DSPG) at T less than Tm. Below a bilayer separation of 100 nm, a repulsive double-layer force is measured, which can be explained through the combined screening and binding effect of the counterions in electrolyte solutions of NaCl, HCl, CaCl2, or mixtures of these. The binding of cations to bilayers in the fluid phase (DMPG) appears to be greater than to bilayers in the gel phase (DSPG). At shorter range, below approximately 3 nm, an attractive interaction is measured in solutions containing CaCl2, which was found to be slightly stronger than the theoretically expected van der Waals interaction. No hydration force was observed to exist in solutions containing CaCl2. In NaCl solutions, the measured interbilayer force can completely be accounted for by the electrostatic repulsion, down to a bilayer separation of at least 2 nm, below which no accurate measurements were possible anymore. Parallel measurements on PG monolayers show that the contraction of a DMPG monolayer following addition of CaCl2 is significantly greater than what is predicted from the change in the double-layer free energy alone. This indicates that changes in the lateral interactions between the lipid headgroups probably involve Ca2+-bridge binding and/or a possible dehydration of the lipid headgroups through Ca2+ binding. The results shed new light on both the interbilayer and intrabilayer interactions of PG and identify the possible factors responsible for the morphological behavior of PG aggregates.     

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements.

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Self-assembly formation of multiple DNA-tethered lipid bilayers

Inspired by natural cell–cell junctions, where membrane-residing proteins control the separation between two or more membranes without interfering with their integrity, we report a new self-assembly route for formation of multiple highly fluid tethered lipid bilayers with the inter-membrane volume geometrically confined by membrane-anchored DNA duplexes. The formation of multiple planar membrane–membrane junctions were accomplished using disk shaped bicelles, composed of a mixture of the long-chained dimyristoyl phosphatidylcholine (DMPC) and the short-chained dihexanoyl PC further stabilized with the positively charged detergent hexadecyl-trimethyl-ammonium bromide (CTAB). Quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) were used to monitor the formation and to characterize the integrity of the self-assembled lipid–DNA architecture.     

Influence of phytosphingosine-type ceramides on the structure of DMPC membrane

The present paper describes the influence of the ceramides with phytosphingosine base, N-stearoylphytosphingosine (Cer[NP]) and alpha-hydroxy-N-stearoylphytosphingosine (Cer[AP]), on the structure and properties of multilamellar (MLVs) and unilamellar vesicles (ULVs) of dimyristoylphosphatidylcholine (DMPC). The lamellar repeat distance, D, has been measured at various temperatures using small angle X-ray diffraction. The incorporation of ceramides into the DMPC membrane causes larger D compared to pure DMPC membrane. For both ceramide types, at 32 degrees C, there is a linear relationship between the D value and the ceramide concentration. However, there is no such dependence at 13 or 60 degrees C. Unlike Cer[AP], Cer[NP] induces a new phase with a repeat distance of 38.5A. The membrane thickness and the vesicle radius of ULVs in water and in sucrose solution were calculated from small angle neutron scattering curves. Phytosphingosine ceramides increase both the membrane thickness and the radius in comparison to pure DMPC ULVs. The stability of ULVs in time was studied by dynamic light scattering. Both ceramides induce an aggregation of the ULVs into micrometer sized non-multilamellar structures in pure water. Presence of sucrose in the environment averts the vesicle aggregation.     

Hisactophilin-mediated binding of actin to lipid lamellae: a neutron reflectivity study of protein membrane coupling.

The neutron reflectivity technique is applied to determine the adsorptive interaction of the 13.5-kDa actin-binding protein hisactophilin from Dictyostelium discoideum with lipid monolayers at a lateral pressure of 21 mN/m < or = pi < or = 25 mN/m at the air-water interface. We compare binding of natural hisactophilin exhibiting a myristic acid chain membrane anchor at the N-terminus (DIC-HIS) and a fatty acid-deficient genetic product expressed in Escherichia coli (EC-HIS). It is demonstrated that only the natural hisactophilin DIC-HIS is capable of mediating the strong binding of monomeric actin to the monolayer, where it forms a layer of about 40 A thickness corresponding to the average diameter of actin monomers. Monolayers composed of pure dimyristoyl phosphatidylcholine with fully deuterated hydrocarbon tails and headgroup (DMPC-d67) and 1:1 mixtures of this lipid with chain deuterated dimyristoyl phosphatidylglycerol (DMPG-d54) are studied on subphases consisting either of fully deuterated buffer (D2O) or of a 9:1 H2O/D2O buffer that matches the scattering length density of air (CMA buffer). The reflectivity data are analyzed in terms of layer models, consisting of one to three layers, depending on the contrast of the buffer and the system. We show that both protein species bind tightly to negatively charged 1:1 DMPC-d67/DMPG-d54 monolayers, thereby forming a thin and most probably monomolecular protein layer of 12-15 A thickness. We find that the natural protein (DIC-HIS) partially penetrates into the lipid monolayer, in contrast to chain-deficient species (EC-HIS), which forms only an adsorbed layer. The coverage of the monolayer with DIC-HIS strongly depends on the presence of anionic DMPG in the monolayer. At a bulk protein concentration of 1.5 micrograms/ml, the molar ratio of bound protein to lipid is about 1:45 for the 1:1 lipid mixture but only 1:420 for the pure DMPC.     

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.     

DMPG gel-fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.