Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Physical properties of insect cuticular hydrocarbons: Model mixtures and lipid interactions

Cuticular lipids include a diverse array of hydrophobic molecules that play an important role in the water economy of terrestrial arthropods. Their waterproofing abilities are believed to depend largely on their physical properties, but little is known about interactions between different surface lipids to determine the phase behavior of the total lipid mixture. I examined the biophysical properties of binary hydrocarbon mixtures, as a model for interactions between different epicuticular lipids of insects. The midpoint of the solid/liquid phase transition (T_m) for mixtures of n-alkanes differing in chain length equaled the weighted average of the T_ms of the component lipids. This was also true for n-alkane-methylalkane mixtures. However, alkane-alkene mixtures melted at temperatures up to 17°C above the temperature predicted from the weighted average of component lipid T_m values. Hydrocarbon mixtures did not exhibit biphasic melting transitions indicative of independent phase behavior of the component lipids. Instead, melting occurred continuously, over a broader temperature range than pure hydrocarbons.     

The binary phase behavior of 1,3-dicaproyl-2-stearoyl-sn-glycerol and 1,2-dicaproyl-3-stearoyl-sn-glycerol

The phase behavior of a binary system constituted of purified 1,3-dicaproyl-2-stearoyl-sn-glycerol (CSC) and 1,2-dicaproyl-3-stearoyl-sn-glycerol (CCS) was investigated at a very slow (0.1 °C/min) and a relatively fast (3.0 °C/min) cooling rate using differential scanning calorimetry (DSC), low resolution NMR, X-ray diffraction (XRD), and polarized light microscopy (PLM). Related forms of the β′ polymorph were detected for all mixtures as well as a β form for CSC-rich mixtures. A double chain length (DCL) stacking of the non-mixed CCS-CCS and CSC-CSC phases and a triple chain length (TCL) stacking of mixed CCS-CSC structure were detected for the different β′ forms. The kinetic phase diagram demonstrated an apparent eutectic at the 0.5_CSC composition when cooled at 0.1 °C/min and at the 0.25_CSC composition when cooled at 3.0 °C/min. The application of a thermodynamic model based on the Hildebrand equation suggests that compounds CSC and CCS are not fully miscible. In addition, the miscibility changes according to the structure of the growing solid phase which is dependent on CSC molar ratio as well as on the kinetics. It was also shown that the miscibility is concentration dependent and that the solid phase, which is growing at conditions well away from equilibrium, is determined kinetically. The molecular interactions were found to be strong and to favor the formation of CSC-CCS pairs in the liquid state. CSC and CCS were also shown to be immiscible in the solid state. Depressions in solid fat content (SFC) were observed for both rates. Relatively complex networks made of needle-like, spherulitic and granular crystals were observed in the CSC/CCS system. A pure CSC phase was found to be instrumental in promoting a higher SFC, and more stable polymorphic forms. The microstructure was shown to be strongly dependent on the cooling rate and was linked to the different polymorphic forms observed by DSC and XRD. Correlations between SFC and the eutectic behavior have been observed for the 3.0 °C/min cooling rate, but not directly in the case of the 0.1 °C/min cooling rate, where slower kinetics which favors the metastable to stable phase conversion processes prevented the same shifts in behavior.     

Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases

The behavior of the two major galactolipids of wheat endosperm, mono- (MGDG) and di-galactosyldiacylglycerol (DGDG) was studied in aqueous dispersion and at the air/liquid interface. The acyl chains of the pure galactolipids and their binary equimolar mixture are in the fluid or liquid expanded phase. SAXS measurements on liquid-crystalline mesophases associated with the electron density reconstructions show that the DGDG adopts a lamellar phase L_α with parallel orientation of the headgroups with respect to the plane of the bilayer, whereas MGDG forms an inverse hexagonal phase H_II with a specific organization of galactosyl headgroups. The equimolar mixture shows a different behavior from those previously described with formation of an Im3m cubic phase. In comparing monolayers composed of the pure galactolipids and their equimolar mixtures, PM-IRRAS spectra show significant differences in the optical properties and orientation of galactosyl groups with respect to the interface. Furthermore, Raman and FTIR spectroscopies show that the acyl chains of the galactolipid mixture are more ordered compared to those of the pure components. These results suggest strong interactions between MGDG and DGDG galactosyl headgroups and these specific physical properties of galactolipids are discussed in relation to their biological interest in wheat seed.     

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.     

Multiple phase equilibria in binary mixtures of phospholipids

Approximate phase diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data presented here, as well as other earlier spin-label, calorimetric, and X-ray data. These phase diagrams represent an improvement over previous studies in that both solid phases (P_β' and L_β') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two P_β' phases that do not form a continuous range of solid solutions.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.     

Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase

The structure and thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2°/min between 25 and 85 °C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 °C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel phase phospholipid to the fluid phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a phase transition of pure phospholipid at about 43 °C coexisting with a liquid-ordered phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 °C which showed that a glucolipid-rich phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 °C with pure phospholipid in gel phase and 42:58 mole ratio at 65 °C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Glass transition behavior of octyl β-D-glucoside and octyl β-D-thioglucoside/water binary mixtures

The lyotropic behavior and glass-forming properties of octyl β-d-glucoside (C8Glu) and octyl β-d-thioglucoside (C8SGlu)/water binary mixtures were evaluated using differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). The results clearly indicate that the mixture forms a glass in the supercooling state of liquid crystalline phases such as cubic, lamellar, and smectic. The glass transition temperature (T_g) of the mixture was strongly dependent on solute concentration, with a higher concentration correlating with a higher T_g. The experimental T_g was consistent with the predicted value calculated using the Couchman-Karasz equation in both the C8Glu and C8SGlu/water mixtures. The change of heat capacity at T_g showed the two bending points under variation of concentrations. And the highest temperature of phase transition from lamellar to isotropic solution was observed at around 50% molar concentration. It was expected that non-percolated state of water existed in extremely higher concentration ranges.     

Phase behaviour of stearic acid-stearonitrile mixtures: A thermodynamic study in bulk and at the air-water interface

The solid-liquid phase behaviour of stearic acid (SA) and stearonitrile (SN) in binary mixtures was investigated by differential scanning calorimetry (DSC), and the formation of SA-SN mixed monolayers at the air-water interface was followed by surface pressure-area (π-A) measurements and by Brewster angle microscope (BAM) observation. The solid-liquid phase diagram is a eutectic type phase diagram, with the eutectic composition 0.90 < X_SN < 0.95 and T^eut = 40.9 °C. The DSC results also suggest that the two components are immiscible in the solid phase but form a liquid mixture with positive deviations to the ideal behaviour. At the air-water interface, the two components form liquid condensed monolayers in the entire range of compositions, at low surface pressures, while solid mixed monolayers only form at high surface pressures for X_SN < 0.8. Thermodynamic analysis indicates that SA and SN are miscible in the liquid condensed phase, with negative deviations from the ideal behaviour. The variation of the collapse surface pressure of mixed monolayers also indicates miscibility at the air-water interface.     

Condensing and fluidizing effects of ganglioside GM1 on phospholipid films

Mixed monolayers of the ganglioside G_M1 and the lipid dipalmitoylphosphatidlycholine (DPPC) at air-water and solid-air interfaces were investigated using various biophysical techniques to ascertain the location and phase behavior of the ganglioside molecules in a mixed membrane. The effects induced by G_M1 on the mean molecular area of the binary mixtures and the phase behavior of DPPC were followed for G_M1 concentrations ranging from 5 to 70 mol %. Surface pressure isotherms and fluorescence microscopy imaging of domain formation indicate that at low concentrations of G_M1 (<25 mol %), the monolayer becomes continually more condensed than DPPC upon further addition of ganglioside. At higher G_M1 concentrations (>25 mol %), the mixed monolayer becomes more expanded or fluid-like. After deposition onto a solid substrate, atomic force microscopy imaging of these lipid monolayers showed that G_M1 and DPPC pack cooperatively in the condensed phase domain to form geometrically packed complexes that are more ordered than either individual component as evidenced by a more extended total height of the complex arising from a well-packed hydrocarbon tail region. Grazing incidence x-ray diffraction on the DPPC/G_M1 binary mixture provides evidence that ordering can emerge when two otherwise fluid components are mixed together. The addition of G_M1 to DPPC gives rise to a unit cell that differs from that of a pure DPPC monolayer. To determine the region of the G_M1 molecule that interacts with the DPPC molecule and causes condensation and subsequent expansion of the monolayer, surface pressure isotherms were obtained with molecules modeling the backbone or headgroup portions of the G_M1 molecule. The observed concentration-dependent condensing and fluidizing effects are specific to the rigid, sugar headgroup portion of the G_M1 molecule.     

The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the ternary system ReLPS/DMPEd54/PLL(500) showed the features of a phase segregation phenomenon not affecting the transition temperatures of the pure lipid components, the ternary system composed of ReLPS/DMPEd54 and OmpF exhibited phase transition curves that were characterized by an unspecific (DMPEd54/OmpF) and a strong and unique (ReLPS/OmpF) type of lipid-protein interaction. Furthermore, semiquantitative estimations supported the supposition that OmpF might be able to induce bilayer asymmetry in preformed symmetrical ReLPS/DMPEd54 vesicles.     

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid “raft” mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 °C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (l_o + s_o) two-phase coexistence region below 8 ± 2 °C at ambient pressure. With increasing temperature, a l_o + l_d + s_o three-phase region is formed, which extends up to ∼27 °C, where a liquid-ordered/liquid-disordered (l_o + l_d) immiscibility region is formed. Finally, above 48 ± 2 °C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, l_d). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the l_o + l_d (+s_o) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 °C, and a pressure range, which extends up to about 2 kbar for T = 37 °C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.     

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: A synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20° to 36 °C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 °C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

Association of a fluorescent amphiphile with lipid bilayer vesicles in regions of solid-liquid-disordered phase coexistence

The effects of solid-fluid phase separations on the kinetics of association of a single-chain fluorescent amphiphile were investigated in two different systems: pure DMPC (dimyristoylphosphatidylcholine) and a 1:1 mixture of DMPC and DSPC (distearoylphosphatidylcholine). In pure DMPC vesicles, solid (s) and fluid (l(d)) phases coexist at the phase transition temperature, T(m), whereas a 1:1 mixture of DMPC and DSPC shows a stable s-l(d) phase separation over a large temperature interval. We found that in single-component bilayers, within the main phase transition, the experimental kinetics of association are clearly not single-exponential, the deviation from that function becoming maximal at the T(m). This observation can be accounted for by a rate of desorption that is slower than desorption from either fluid or solid phases, leaving the rates of insertion unchanged, but a treatment in terms of stable fluid and solid domains may not be adequate for the analysis of the association of an amphiphile with pure DMPC vesicles at the T(m). In DMPC/DSPC mixtures with solid-fluid phase coexistence, association occurs overall faster than expected based on phase composition. The observed kinetics can be described by an increase in the rate of insertion, leaving the desorption rates unchanged. The fast kinetics of insertion of the amphiphile into two-phase bilayers in two-component vesicles is attributed to a more rapid insertion into defect-rich regions, which are most likely phase boundaries between solid and fluid domains. A two-component mixture of lipids that shows a stable phase separation between l(d)-s phases over a large temperature interval thus behaves very differently from a single-component bilayer at the T(m), with respect to insertion of amphiphiles.     

Membrane domain formation, interdigitation, and morphological alterations induced by the very long chain asymmetric C24:1 ceramide

Ceramide (Cer) is involved in the regulation of several biological processes, such as apoptosis and cell signaling. The alterations induced by Cer in the biophysical properties of membranes are thought to be one of the major routes of Cer action. To gain further knowledge about the alterations induced by Cer, membrane reorganization by the very long chain asymmetric nervonoylceramide (NCer) was studied. The application of an established fluorescence multiprobe approach, together with x-ray diffraction, differential scanning calorimetry, and confocal fluorescence microscopy, allowed the characterization of NCer and the determination of the phase diagram of palmitoyloleoylphosphatidylcholine (POPC)/NCer binary mixtures. Nervonoylceramide undergoes a transition from a mixed interdigitated gel phase to a partially interdigitated gel phase at ∼20°C, and a broad main transition to the fluid phase at ∼52°C. The solubility of NCer in the fluid POPC is low, driving gel-fluid phase separation, and the binary-phase diagram is characterized by multiple and large coexistence regions between the interdigitated gel phases and the fluid phase. At 37°C, the relevant phases are the fluid and the partially interdigitated gel. Moreover, the formation of NCer interdigitated gel phases leads to strong morphological alterations in the lipid vesicles, driving the formation of cochleate-type tubular structures.     

Solid Solution Domains at Phase Transition Front of LixNi0.5Mn1.5O4

Nickel‐substituted manganese spinel LiNi_0.5Mn_1.5O₄ (LNMO) is a promising 5 V class positive electrode material for lithium‐ion batteries. As micron‐sized LNMO particles show high rate capability in its two‐phase coexistence regions, the phase transition mechanism is of great interest in understanding the electrode behavior at high rates. Here, the phase transition dynamics of Li_xNi_0.5Mn_1.5O₄ is elucidated on high rate charging–discharging using operando time‐resolved X‐ray diffraction (TR‐XRD). The TR‐XRD results indicate the existence of intermediate states, in addition to the thermodynamically stable phases, and it is shown that the origin of such intermediate states is ascribed to the solid‐solution domains at the phase transition front, as supported by the analysis using transmission electron microscopy coupled with electron energy‐loss spectroscopy. The phase transition pathways dependent on the reaction rate are shown, together with possible explanation for this unique transition behavior.     

Phase fluctuations on the micron-submicron scale in GUVs composed of a binary lipid mixture

We used a combination of imaging and fluctuation techniques to investigate the temporal evolution of gel phase domains at the onset of phase separation, as well as the correlation between domain topology and local lipid ordering in GUVs composed of a binary mixture of DPPC/DLPC 1:1. The data acquired at temperatures immediately above the transition temperature of the two lipids suggest fluctuations in the lipid organization with a lifetime <0.1 s and a characteristic length of 1.2 μm. As the temperature is decreased below the transition temperature of one of the lipids, coupling between the two leaflets of the bilayer is observed to begin within the first five minutes after the onset of phase separation. However, domains confined to only one leaflet can be found during the first 45-50 min after the onset of phase separation. Our analysis using a two-state model (liquid and gel) indicates that for the first 45-50 min from the onset of phase separation the two lipid phases do not strongly influence the phase behavior of each other on the micron-length scale. At longer times, behavior that deviates from the two-state model is observed and appears to be correlated to domain morphology.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: II. On the fine structure of the phase transitions in hydrated dipalmitoylphosphatidylethanolamine

The phase transitions of dipalmitoylphosphatidylethanolamine (DPPE) in excess water have been examined by low-angle time-resolved x-ray diffraction and calorimetry at low scan rates. The lamellar subgel/lamellar liquid-crystalline (L_c → L_α), lamellar gel/lamellar liquid-crystalline (L_β → L_α), and lamellar liquid-crystalline/lamellar gel (L_α → L_β) phase transitions proceed via coexistence of the initial and final phases with no detectable intermediates at scan rates 0.1 and 0.5°C/min. At constant temperature within the region of the L_β → L_α transition the ratio of the two coexisting phases was found to be stable for over 30 min. The state of stable phase coexistence was preceded by a 150-s relaxation taking place at constant temperature after termination of the heating scan in the transition region. While no intermediate structures were present in the coexistence region, a well reproducible multipeak pattern, with at least four prominent heat capacity peaks separated in temperature by 0.4-0.5°C, has been observed in the cooling transition (L_α → L_β) by calorimetry. The multipeak pattern became distinct with an increase of incubation time in the liquid-crystalline phase. It was also clearly resolved in the x-ray diffraction intensity versus temperature plots recorded at slow cooling rates. These data suggest that the equilibrium state of the L_α phase of hydrated DPPE is represented by a mixture of domains that differ in thermal behavior, but cannot be distinguished structurally by x-ray scattering.