Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures. Structural changes between hexagonal, cubic and bilayer phases

The polymorphic phase behaviour of dilinoleoylphosphatidyethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is investigated by freeze-fracture electron microscopy, X-ray diffraction and 31P-NMR. The structures at 5% or less POPC are predominantly inverted hexagonal (HII), whereas at 15% or more POPC, the structure is mostly bilayer (L), interrupted by defects (lipidic particles). A cubic phase structure is observed in the transition range between H and L phases; the cubic arrangement deteriorates at higher temperatures into an amorphous aggregate of spherical units. Both cubic and amorphous structures contribute to the isotropic 31P resonance, with no preference for PC or PE partitioning in the isotropic motion as observed by high resolution NMR. The existence of the cubic phase seems to depend cirtically on the homogeneity and the degree unsaturation of the phospholipids.     

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal amphiphile phases. III. Isotropic and inverted cubic state formation via intermediates in transitions between L alpha and HII phases

Inverted cubic and isotropic phases have been observed in phospholipid and glycolipid systems. These phases exhibit characteristic morphologies in freeze-fracture electron micrographs, isotropic 31P-NMR resonances and (in some cases) cubic X-ray diffraction patterns. It is proposed here that these phases may form from the same intermediates that are involved in lamellar/inverted hexagonal (L alpha/HII) phase transitions, and that it is possible that these cubic and isotropic phases are metastable. According to a kinetic theory of L alpha/HII phase transitions, intermediates in such transitions can form structures known as interlamellar attachments (ILAs). It is shown that ILAs should form in large numbers during L alpha/HII transitions in systems like those reported to form inverted cubic or isotropic structures. ILAs cannot readily assemble into either the HII phase or well-ordered arrays of L alpha phase bilayers, and represent a kinetic trap for intermediates in L alpha/HII transitions (although it is possible that they are marginally more stable in a thermodynamic sense than the L alpha phase in a small temperature range below TH). It is also shown that arrays of ILAs should form metastable arrays with the same morphology and isotropic 31P-NMR resonances that are observed in isotropic and inverted cubic states. In particular, under some circumstances ILAs will assemble into a structure identical to the bicontinuous inverted cubic phase previously described in monoglycerides and very similar in morphology to structures observed in phospholipid systems. Finally, since isotropic and cubic states form from ILAs, which also can mediate fusion of unilamellar vesicles, unilamellar vesicles should fuse to at least some extent under the same conditions in which multilamellar samples of the same lipid form isotropic or inverted cubic states. This correlation has been observed.     

Lipid Polymorphism Induced by Surfactant Peptide SP-B1-25

Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B_1-25, a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B_1-25 interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B_1-25 on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B_1-25 partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B_1-25 remains constant, but ²H and ³¹P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T_m of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B_1-25, a return to a single lamellar phase above the lipid mixture T_m is observed, but for 5 mol% SP-B_1-25 a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in ²H and ³¹P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B_1-25 promotes the formation of a fluid isotropic phase. The ability of SP-B_1-25 to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of SP-B_8-25 and SP-B_59-80, indicating a critical role for the proline rich first seven amino acids in this protein.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

The Effect of a C-Terminal Peptide of Surfactant Protein B (SP-B) on Oriented Lipid Bilayers, Characterized by Solid-State H- and P-NMR

SP-B_CTERM, a cationic, helical peptide based on the essential lung surfactant protein B (SP-B), retains a significant fraction of the function of the full-length protein. Solid-state ²H- and ³¹P-NMR were used to examine the effects of SP-B_CTERM on mechanically oriented lipid bilayer samples. SP-B_CTERM modified the multilayer structure of bilayers composed of POPC, POPG, POPC/POPG, or bovine lipid extract surfactant (BLES), even at relatively low peptide concentrations. The ³¹P spectra of BLES, which contains ∼1% SP-B, and POPC/POPG with 1% SP-B_CTERM, look very similar, supporting a similarity in lipid interactions of SP-B_CTERM and its parent protein, full-length SP-B. In the model systems, although the peptide interacted with both the oriented and unoriented fractions of the lipids, it interacted differently with the two fractions, as demonstrated by differences in lipid headgroup structure induced by the peptide. On the other hand, although SP-B_CTERM induced similar disruptions in overall bilayer orientation in BLES, there was no evidence of lipid headgroup conformational changes in either the oriented or the unoriented fractions of the BLES samples. Notably, in the model lipid systems the peptide did not induce the formation of small, rapidly tumbling lipid structures, such as micelles, or of hexagonal phases, the observation of which would have provided support for functional mechanisms involving peptide-induced lipid flip-flop or stabilization of curved lipid structures, respectively.     

Cubic topology in surfactant and lipid mixtures

Ternary systems of palmitoyl-oleoyl-phosphatidylcholine (POPC) and the non-ionic surfactant C₁₂EO₂ (di-ethylene-oxide-mono-dodecyl-ether) in water have been studied with optical microscopy, NMR, DSC and X-rays from ambient temperatures to 45 °C. Below 29 °C the system is in the lamellar liquid crystalline state. Between 30 and 32 °C it transforms into a cubic Ia3d structure which converts into the cubic Pn3m phase at 39 °C. The transitions are fully reversible. An epitaxial relationship between all three phases was found, which is an elegant and convenient way to rearrange molecules from lamellar bilayers to a network of curved surfaces. The la3d (Q²³⁰) to Pn3m (Q²²⁴) transition occurs without measurable enthalpy change. This, together with the metric relation of 1.60 between the cubic lattice constants is strong evidence for a Bonnet transformation, where the structural changes occur without change in curvature. The potential significance of the cubic phases as intermediate structures for biological processes, e. g. transport across a bilayer or fusion of membranes, are discussed.     

Interaction of the pitavastatin with model membranes

Pitavastatin is a statin drug that, by competitively inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase, can lower serum cholesterol levels of low-density lipoprotein (LDL) accompanied by side effects due to pleiotropic effects leading to statin intolerance. These effects can be explained by the lipophilicity of statins, which creates membrane affinity and causes statin localization in cellular membranes. In the current report, the interaction of pitavastatin with POPC model membranes and its influence on the membrane structure were investigated using ¹H, ²H and ³¹P solid-state NMR spectroscopy. Our experiments show the average localization of pitavastatin at the lipid/water interface of the membrane, which is biased towards the hydrocarbon core in comparison to other statin molecules. The membrane binding of pitavastatin also introduced an isotropic component into the ³¹P NMR powder spectra, suggesting that some of the lamellar POPC molecules are converted into highly curved structures.     

Detergent-like properties of magainin antibiotic peptides: A P solid-state NMR spectroscopy study

³¹P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in ³¹P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid ³¹P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type ³¹P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Melatonin Alters Fluid Phase Coexistence in POPC/DPPC/Cholesterol Membranes

The structure and biophysical properties of lipid membranes are important for cellular functions in health and disease. In Alzheimer’s disease, the neuronal membrane is a target for toxic amyloid-β (Aβ). Melatonin is an important pineal gland hormone that has been shown to protect against Aβ toxicity in cellular and animal studies, but the molecular mechanism of this protection is not fully understood. Melatonin is a small membrane-active molecule that has been shown to interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components. To make it more relevant to neuronal membranes, we used a more complex ternary lipid mixture as our membrane model. In this study, we used ²H-NMR to investigate the effect of melatonin on the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol lipid membranes. We used deuterium-labeled POPC-d₃₁ and DPPC-d₆₂,separately to probe the changes in hydrocarbon chain order as a function of temperature and melatonin concentration. We find that POPC/DPPC/cholesterol at molar proportions of 3:3:2 is close to liquid-disordered/liquid-ordered phase separation and that melatonin can induce phase separation in these ternary mixtures by preferentially incorporating into the disordered phase and increasing its level of disorder. At 5 mol% melatonin, we observed phase separation in samples with POPC-d₃₁, but not with DPPC-d₆₂, whereas at 10 mol% melatonin, phase separation was observed in both samples with either POPC-d₃₁ or DPPC-d₆₂. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against Aβ, and that choice of chain perdeuteration is an important consideration from a technical point of view.     

Further evidence for closed, nonspherical aggregates in the cubic I1 phase of lysolecithin and water

Measurements of time-resolved fluorescence quenching have been performed in the binary lauroyllysophosphatidylcholine (LaLPC)/water system. The aggregation numbers, N, are determined for the micellar solution phase (N_micelle ≈ 80) and the cubic liquid crystalline I₁ phase (N_cub ≈ 90) at 298-303 K. When a quencher is present, the fluorescence decays for the hexagonal phase of the LaLPC/water system and for the bicontinuous cubic phase of monooleoylglycerol/water system are nonexponential, as expected for phase structures having long-range continuous apolar regions. Nuclear magnetic resonance (NMR) measurements of the lipid translational diffusion conclusively show that the cubic I₁ phase consists of closed micelles. NMR spectra of ³¹P obtained at 202.4 MHz of this cubic phase exhibit a characteristic line shape, which is compatible with a phase structure containing short nonspherical micelles. A comparison between electron spin resonance (ESR) spin-label spectra recorded for a micellar solution and the cubic phases of the LaLPC and monooleoylglycerol systems are also shown to support a structure of closed micelles in the cubic I₁ phase of the lysolecithin system.     

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C₆-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by ²H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C₆-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel?+?fluid phase coexistence. Inclusion of both fluorescent probes (1?mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C₆-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.     

Membrane fusion and inverted phases

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.     

DHA Modifies the Size and Composition of Raftlike Domains: A Solid-State 2H NMR Study

Docosahexaenoic acid is an omega-3 polyunsaturated fatty acid that relieves the symptoms of a wide variety of chronic inflammatory disorders. The structural mechanism is not yet completely understood. Our focus here is on the plasma membrane as a site of action. We examined the molecular organization of [²H₃₁]-N-palmitoylsphingomyelin (PSM-d₃₁) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane by solid-state ²H NMR. The spectra were analyzed in terms of segregation into ordered SM-rich/chol-rich (raftlike) and disordered PC-rich/chol-poor (nonraft) domains that are nanoscale in size. An increase in the size of domains is revealed when POPC was replaced by PDPC. Spectra that are single-component, attributed to fast exchange between domains (<45 nm), for PSM-d₃₁ mixed with POPC and chol become two-component, attributed to slow exchange between domains (r > 30 nm), for PSM-d₃₁ mixed with PDPC and chol. The resolution of separate signals from PSM-d₃₁, and correspondingly from [3α-²H₁]cholesterol (chol-d₁) and 1-[²H₃₁]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d₃₁), in raftlike and nonraft domains enabled us to determine the composition of the domains in the PDPC-containing membrane. Most of the lipid (28% SM, 29% chol, and 23% PDPC with respect to total lipid at 30°C) was found in the raftlike domain. Despite substantial infiltration of PDPC into raftlike domains, there appears to be minimal effect on the order of SM, implying the existence of internal structure that limits contact between SM and PDPC. Our results suggest a significant refinement to the model by which DHA regulates the architecture of ordered, sphingolipid-chol-enriched domains (rafts) in membranes.     

Induction of a hexagonal phase in phospholipid-surfactant bilayers

The possibility of modifying the curvature of lipid bilayers by mixing them with additives is demonstrated and the evolution of geometrical parameters with composition is discussed. X-ray diffraction patterns of the POPC/C₁₂EO₂/²H₂O system were observed as a function of the relative humidity. The formation of an unexpected hexagonal phase indicates peculiar behaviour in these mixtures. The cylinder radius of this phase is considerably smaller than previously observed in cubic phases. A discussion of the head group interactions is presented. We have also been able to show that the uptake of water by the L _β gel phase is higher as a single phase than in the L _β +H_II two phase region. The water content is important for the stabilization of H_II phase and determination of its characteristic dimensions. However, it is argued that the interaction between the surfactant and the lipid is the key factor for its formation and that the EO₂ head groups displace water from the inner parts of the polar region of the mesogenic units. Received: 22 September 1997 / Revised version: 31 December 1997 / Accepted: 10 February 1998     

Structural Polymorphisms of Rough Mutant Lipopolysaccharides Rd to Ra from Salmonella minnesota

The structural polymorphisms of rough mutant lipopolysaccharides (LPS) Rd, Rc, Rb, and Ra from Salmonella minnesota (strains R4, R7, Rz, R5, R345, and R60, respectively) were investigated as a function of temperature, water content, and Mg²⁺ concentration. The gel to liquid crystalline (B↔α) phase transition temperature (T_c) and the state of order within each phase were measured by Fourier transform infrared spectroscopy. The amount of bound water was determined by differential scanning calorimetry and the three-dimensional structures in each phase state were characterized by synchrotron radiation X-ray diffraction. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The B↔α acyl chain melting temperatures at high water contents (95-97%), T_c = 31 to 32°C for LPS Rd, 33 to 35°C for LPS Rc to Rb, and 36°C for LPS Ra, increase with decreasing water content and in the presence of Mg²⁺ cations with a concomitant broadening of the transition range. Below 30 to 50% water content, no distinct phase transitions can be observed. These effects are most pronounced for LPS with the shortest sugar chains. Below 50% water content, only lamellur structures can be observed in the temperature range 5 to 80°C, independent of the Mg²⁺ concentration. Above 50% water concentration, for large [LPS]:[Mg²⁺] molar ratios the predominant structure above T_c is nonlamellar; for smaller [LPS]:[Mg²⁺] molar ratios a superposition of lamellar and nonlamellar structures is found. For all LPS Rd to Rb at low Mg²⁺ concentrations, the phase transition is connected with a change in the three-dimensional structure from lamellar or mixed lamellar/nonlamellar to a pure nonlamellar, probably cubic structure. The tendency to form nonlamellar structures decreases with the length of the core oligosaccharide. At an equimolar ratio of [LPS] and [Mg²⁺] a multibilayered organization is observed. Some of the nonlamellar structures are cubic phases with periodicities between 12 and 16 nm. The molecular dimensions of the single endotoxin molecules in the absence and the presence of external water are estimated from the different lamellar periodicities, including those of free lipid A and deep rough mutant LPS Re. These observations are discussed with respect to the biological importance of LPS as a potent inducer of biological effects in mammals.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Effects of Lipid Composition and Phase on the Membrane Interaction of the Prion Peptide 106-126 Amide

Lipid rafts are specialized liquid-ordered (L_o) phases of the cell membrane that are enriched in sphingolipids and cholesterol (Chl), and surrounded by a liquid-disordered (L_d) phase enriched in glycerophospholipids. Lipid rafts are involved in the generation of pathological forms of proteins that are associated with neurodegenerative diseases. To investigate the effects of lipid composition and phase on the generation of pathological forms of proteins, we constructed an L_d-gel phase-separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (from bovine brain (BSM))-supported lipid bilayer (SLB) and an L_d-L_o phase-separated POPC/BSM/Chl SLB. We used in situ time-lapse atomic force microscopy to study the interactions between these SLBs and the prion peptide K¹⁰⁶TNMKHMAGAAAAGAVVGGLG¹²⁶ (PrP106-126) amide, numbered according to the human prion-peptide sequence. Our results show that: 1), with the presence of BSM in the L_d phase, the PrP106-126 amide induces fully penetrated porations in the L_d phase of POPC/BSM SLB and POPC/BSM/Chl SLB; 2), with the presence of both BSM and Chl in the L_d phase, the PrP106-126 amide induces the disintegration of the L_d phase of POPC/BSM/Chl SLB; and 3), with the presence of both BSM and Chl in the L_o phase, PrP106-126 amide induces membrane thinning in the L_o phase of POPC/BSM/Chl SLB. These results provide comprehensive insight into the process by which the PrP106-126 amide interacts with lipid membranes.     

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the ²H NMR spectra of 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.     

Characterization of the ternary mixture of sphingomyelin, POPC, and cholesterol: support for an inhomogeneous lipid distribution at high temperatures

A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, ²H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by ²H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger ²H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.