Lamellar Repeat Research Articles

Effect of Confinement on Membrane Undulation in a Swollen Lamellar Phase

We have investigated a static structure and membrane undulation in a swollen lamellar phase by small-angle neutron scattering and neutron spin echo (NSE). The effective viscosity, estimated by NSE data analysis using Zilman and Granek's theory [Phys. Rev. Lett. 77 (1996) 4788], was larger than the bare viscosity of the solvent, and increased with decreasing lamellar repeat distance. A theory given by Gov et al. [Phys. Rev. E 70 (2004) 011104] for hydrodynamics in confined fluid membranes could explain the tendency of NSE results. This indicates that the interference from neighbouring membranes neglected in Zilman and Granek's theory should be considered in analyzing the membrane dynamics in the swollen lamellar phase from the NSE data.

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A 2. Thus the amide phospholipids l-ether-amide-PC (1- O-hexadecyl-2- N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), l-ester-amide-PC (1-palmitoyl-2- N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and l-ether-amide-PE (1- O-hexadecyl-2- N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 °C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for l-ester-amide-PC and l-ether-amide-PC, whereas the temperature dependence is opposite for the l-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.

Morphology Development of Ultrathin Symmetric Diblock Copolymer Film via Solvent Vapor Treatment

We have followed the time development of the microdomain structure in symmetric diblock copolymer poly(styrene-b-methyl methacrylate), P(S-b-MMA), ultrathin films via PMMA-selective solvent vapor treatment by atomic force microscopy (AFM). After preparation on a substrate preferentially attracting the PMMA block, PS forms a continuous layer at a film's free surface. With subsequent solvent vapor treatment, the film gradually shows a well-ordered hexagonally packed nanocylinders structure. It is shown that only when the film thickness is less than the 1/2L0 (lamellar repeat spacing), and exposed to PMMA block selective solvent for an appropriate time, can the well-ordered hexagonally packed nanocylinders form. On an extended solvent vapor treatment, a mixed morphology containing nanocylinders and stripes appears, followed by the striped morphologies. When the annealing time is long enough, the film comes back to the flat surface again, however, with PMMA instead of PS dominating the free surface. Thickness confinement and solvent induced reconstruction of the film are shown to be responsible for the P(S-b-MMA) morphology and surface chemistry development.

Structure and fluctuations of phosphatidylcholines in the vicinity of the main phase transition.

We have determined the structural properties and bending fluctuations of fully hydrated phosphatidylcholine multibilayers in the fluid (Lalpha) phase, as well as the structure of the ripple (Pbeta') phase near the main phase transition temperature (TM) by x-ray diffraction. The number of carbons, nHC, per acyl chain of the studied disaturated lipids varied from 14 to 22. All lipids exhibit a nonlinear increase of the lamellar repeat distance d in the Lalpha phase upon approaching TM, known as "anomalous swelling." The nonlinear increase reduces with chain length, but levels off at a constant value of about 0.5 A for lipids with more than 18 hydrocarbons per chain. A detailed analysis shows that anomalous swelling has two components. One is due to an expansion of the water layer, which decreases with chain length and finally vanishes for nHC >18. The second component is due to a bilayer thickness increase, which remains unchanged in its temperature dependence, including a nonlinear component of about 0.5 A in the vicinity of TM. Thus, anomalous swelling above 18 hydrocarbons per chain is due to the pretransitional effects on the membrane only. These results are supported by a bending fluctuation analysis revealing increased undulations close to TM only for the short chain lipids. We have further calculated the electron density maps in the ripple phase and find no coupling of the magnitude of the ripple amplitude to the chain length effects observed in the Lalpha phase. Hence, in agreement with an earlier report by Mason et al. [Phys. Rev. E 63, 030902 (2001)] there is no connection between the formation of the ripple phase and anomalous swelling.

Columnar DNA Superlattices in Lamellar o-Ethylphosphatidylcholine Lipoplexes: Mechanism of the Gel-Liquid Crystalline Lipid Phase Transition

DNA arranges into rectangular columnar superlattices between interdigitated lipid bilayers in the low-temperature gel phase of the lipoplexes of the cationic o-ethylphosphatidylcholines. The interlamellar correlation in the DNA ordering is not observed within the liquid crystalline lipid phase. The gel−liquid crystalline phase transition of the lipid induces a contraction of the DNA smectic array that matches the increased positive charge density of the membrane surface caused by the interdigitated−noninterdigitated transition. The transition proceeds via an intermediate state that has an expanded lamellar repeat period.

Finite-size effects do not reduce the repeat spacing of phospholipid multibilayer stacks on a rigid substrate.

Finite-size effects in stacks of phospholipid bilayers, in the fluid L alpha phase, are investigated using samples oriented on silicon substrates. Recently in this journal, such effects have been suggested as the probable cause of reduced lamellar repeat spacings in very thin samples made up of a few (<10) bilayers. Our systematic studies on samples of different thicknesses do not support this conclusion. At full hydration all samples are found to have the same repeat spacing, irrespective of their thickness. At lower hydrations, on the other hand, very thin samples, consisting of only a few bilayers, have a slightly larger spacing.

Phase behavior of dipalmitoylphosphatidylethanolamine in dimethylsulfoxide–water mixture

The phase behaviors of dipalmitoylphosphatidylethanolamine (DPPE) dispersed in dimethyl sulfoxide (DMSO) aqueous solutions were studied by using differential scanning calorimetry and X-ray diffraction methods. At low concentration of DMSO (mole fraction, x<0.2), phase transition between lamellar-gel and lamellar liquid-crystal phase can be observed during heating/cooling cycles at a scanning rate of 5 °C min −1. The formation of the stable lamellar-crystal phase from the metastable lamellar-gel phase needs long time incubation. At moderate concentration of DMSO (0.2< x<0.37), phase transition from lamellar-gel to crystalline phase can be detected during either cooling or heating at the same scanning rate. At high concentrations ( x>0.37), DMSO promotes remarkably the formation of lamellar-crystal phase of the lipid, resulting in phase transitions between lamellar-crystal phase and liquid-crystal phase during heating/cooling cycling. A phase diagram has been constructed over the entire concentration range of the mixed solvent. In addition, it was found that DMSO could decrease the lamellar repeat spacing of crystalline and liquid crystalline phase.

Phase Segregation in Gradient Copolymer Melts

Local segregation in melts of copolymers with composition gradients along their backbones is analyzed. The transition to a lamellar periodic structure as the effective degree of incompatibility χN increases is studied for symmetric copolymers with various composition gradients. A numerical self-consistent mean-field (SCMF) technique is used to characterize the ordered lamellar state in the weak and strong segregation regimes, and the random phase approximation (RPA) is used to calculate the scattering function analytically and find the location of the critical order−disorder transition for each melt. The critical point increases from (χN)c = 10.495 for block copolymers to (χN)c = 29.25 for a fully tapered linear gradient copolymer. For broad composition gradients the equilibrium lamellar repeat length is shorter for a given value of χN, and the unit cell composition profile is more sinusoidal. The dependence of the equilibrium repeat distance on χN is nearly universal when renormalized by the critical point of each copolymer.

The Effect of Fructan on the Phospholipid Organization in the Dry State

Fructans, a family of oligo- and polyfructoses, are implicated to play a drought-protecting role in plants. Inulin-type fructan is able to preserve the membrane barrier during dehydration. However, whether other fructans would be able to perform this function is unknown. In addition, almost nothing is known about the organization of these systems, which could give insight into the protective mechanism. To get insight into these questions the effect of different fructans on phosphatidylcholine-based model systems under conditions of dehydration was analyzed. Using a vesicle leakage assay, it was found that both levan- and inulin-type fructans protected the membrane barrier. This suggests that fructans in general would be able to protect the membrane barrier function. Furthermore, both fructan-types inhibited vesicle fusion to a large extent as measured using a lipid-mixing assay. Using x-ray diffraction, it was found that in the presence of both inulin- and levan-type fructans the lamellar repeat distance increased considerably. From this it was concluded that fructans are present between the lipid bilayers during drying. Furthermore, they stabilize the L α phase. In contrast to fructans, dextran did not increase the lamellar repeat distance and it even promoted L β phase formation. These data support the hypothesis that fructans can have a membrane-protecting role during dehydration, and give insight into the mechanism of protection.

Organization of Skin Stratum Corneum Extracellular Lamellae: Diffraction Evidence for Asymmetric Distribution of Cholesterol

Lipid suspensions containing 2:1:1 skin ceramides:palmitic acid:cholesterol, similar to the lipid composition found in the extracellular matrix of skin stratum corneum, were analyzed by X-ray diffraction methods. These suspensions gave a sharp wide-angle reflection at 4.1Å, indicating tight hydrocarbon chain packing that would function as a water barrier, and low-angle lamellar diffraction with a repeat period near 130Å, similar to that previously recorded from intact stratum corneum. The lamellar repeat increased from 121Å at pH 6 to 133Å at pH 8.5, allowing phase angles of the lamellar data to be obtained by a sampling theorem “swelling” analysis. Electron density profiles showed that each repeating unit contained two asymmetric bilayers, with a fluid space on one side of the bilayer that increased with increasing pH, due to electrostatic repulsion between bilayers because of ionization of the palmitic acid. Profiles obtained from lamellae with cholesterol sulfate partially substituted for cholesterol showed large density increases on that same side of the bilayer, indicating that cholesterol is asymmetrically distributed in each bilayer. A molecular model was developed postulating that this asymmetry is due to the exclusion of cholesterol from lipid monolayers containing the ester-linked unsaturated (linoleic) hydrocarbon chain of skin ceramide 1. This model can explain the altered organization of extracellular lamellae in epidermal cysts (P. W. Wertz, D. C. Swartzendruber, K. C. Madison, D. T. Downing. 1987. J. Invest. Dermatol. 89:419–425) where the ester-linked chains have a higher percentage of saturated fatty acids than found in normal epidermis.

Structure and Conformation of Bipolar Tetraether Lipid Membranes Derived from Thermoacidophilic Archaeon Sulfolobus acidocaldarius as Revealed by Small-Angle X-ray Scattering and High-Pressure FT-IR Spectroscopy

The phase behavior, conformation, and structure of bipolar tetraether liposomes composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius have been studied by small-angle X-ray scattering (SAXS) and high-pressure Fourier transform infrared spectroscopy (FT-IR) at pD 2.15. The SAXS data on PLFE multilamellar vesicles in 85 wt % D 2 O showed two orders of lamellar Bragg reflections over the temperature range 5-75 °C. The lamellar repeat distances d indicate three lamellar regions: 5-50 °C (d, ∼49-50 A), 50-60 °C (∼50-54 A), and 60-74 °C (∼54-56 A). The 4-A increase in d-spacing from 50 to 60 °C is probably due to an increase in hydration at the PLFE polar headgroups and/or a stretch of the polar headgroups toward the bulk aqueous phase. At 74-75 °C, a lamellar-to-cubic phase transition occurs. The reciprocal spacings of the cubic phases correspond to the coexistence of the inverse bicontinuous cubic phases Q I I D and Q I I P with lattice constants of 105 and 82 A, respectively. At atmospheric pressure, the vibrational frequency of the CH 2 symmetric stretching mode showed transitions at ∼46-48, 61, and 74 °C, in agreement with the SAXS data. The overall 2 cm - 1 increase in wavenumber between 61 and 74 °C indicates a transition from a rigid, gellike lipid conformation to a chain conformation with considerable disorder, as expected from cubic phases which appear above ∼74 °C. The pressure dependence (up to 15 kbar) of the symmetric CH 2 stretching vibrational wavenumber at various temperatures has also been examined. A variety of new gellike phases are observed at elevated pressures, showing again a rich polymorphism in PLFE liposomes. The most prominent pressure-induced transition involves a ∼2 cm - 1 increase in wavenumber, which appears at 8.0 kbar at 60 °C, 8.4 kbar at 43 °C, and 10.8 kbar at 20 °C, giving an unusual negative dT/dp value. The possible source of this anomaly has been discussed in terms of the temperature attenuation of the hydrogen bond network in the polar headgroup region.

Phase behavior of charged lipid bilayer membranes with added electrolyte

We investigate the phase behavior of the aqueous solution of charged lipid bilayer membranes forming a lamellar structure in the presence of the added electrolyte. The Goldstein–Leibler model [Phys. Rev. A 40, 1025 (1989)] proposed for neutral lipids is extended by taking into account the screened electrostatic interaction which belongs to the so called intermediate region of the Poisson–Boltzmann theory. We mainly focus on the variation of the phase behavior as the added electrolyte concentration is changed. By decreasing the electrolyte concentration, the electrostatic repulsion between the neighboring membranes becomes stronger. As a result, the maximum equilibrium swelling composition shifts to larger water content, and the lamellar repeat distance increases although the membrane thickness remains almost constant. Our results recover not only the experimentally observed phase diagram for the charged lipid, but also the dependence of the lamellar repeat distance both on the electrolyte concentration and on the lipid composition. Some discussions are provided for the case when the electrostatic interaction is not screened due to the absence of any electrolyte.

Phase behavior in multilamellar vesicles of DPPC containing ganglioside GM3 with a C18:1 sphingoid base and a 24:0 acyl chain (GM3(18,24)) observed by X-ray diffraction

Structures and phase behavior of multilamellar vesicles of 1,2-dipalmitoyl- l-phosphatidylcholine (DPPC) containing various amount of ganglioside GM3 with a C18:1 sphingoid base and a 24:0 acyl chain (GM3(18,24)) were investigated by small-angle X-ray diffraction. Below 3.5 mol% GM3 content, the phase behavior was similar to that of pure DPPC except for a slight increase of lamellar repeat distance in the L β′, the P β′ and the L α phases and a decrease of the pretransition temperature. In the range of 4–12 mol% GM3 content, another phase which has larger repeat distances coexisted with the phase observed below 3.5 mol% GM3 content. This has been interpreted that the phase separation into GM3-poor phase (denoted as A-phase) and GM3-rich phase (denoted as B-phase) took place. Above 13 mol% GM3 content, the B-phase became dominant. This phase separation may be related to the formation of GM3-enriched microdomains that had been observed on the cell surfaces which express large amounts of GM3, such as murine B16 melanoma (J. Biol. Chem. 260 (1985) 13328).

Structure and Interactions in the Anomalous Swelling Regime of Phospholipid Bilayers

We have carried out X-ray and neutron diffraction experiments, as a function of temperature, on fully hydrated samples of dimyristoyl phosphatidylcholine bilayers. The data show the following: (a) In the vicinity of the Lα to Pβ‘ transition, we find an anomalous expansion of the water layer of ∼1.7 A. (b) The lipid bilayer thickness increases quasi-linearly and is similar to the increase in the lamellar repeat spacing d found in dimyristoyl ethanolamine bilayers in the temperature range of TM to TM + 13 °C. (c) In contrast to an earlier study, we find no significant changes to the steric size of the phosphatidylcholine headgroup. The anomalous increase in d is thus dominated by an expansion of the water layer. This expansion is caused by a distinct increase in bilayer fluctuations as revealed by an analysis of the Caille parameter. Additional osmotic pressure experiments not only support this notion but have allowed us to further estimate the temperature dependence of both the bilayer bending rigidity, K...

The interaction of α-tocopherol with bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine

The effect of α-tocopherol on the structure and phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylcholine was examined by real-time synchrotron X-ray diffraction and freeze-fracture electron microscopic methods. X-ray scattering intensity was recorded from mixed aqueous dispersions of phospholipid with 2.5, 5, 10 and 20 mol% α-tocopherol during temperature scans at 3°/min between −25 and 10 °C. A ripple structure is induced by the presence of α-tocopherol that coexists with the ripple phase characteristic of the pure phospholipid in mixtures containing 2.5 mol% α-tocopherol but completely replaces it in mixtures containing greater proportions of α-tocopherol. Freeze-fracture replicas of dispersions containing 5 mol% α-tocopherol indicate a ripple phase with a periodicity of about 9 nm. Increasing amounts of α-tocopherol result in a progressive reduction in temperature of the gel to liquid–crystal phase transition and broadening of the transition. Two lamellar phases coexist in the liquid–crystal state, one with a spacing of 6.4 nm assigned to an α-tocopherol-enriched lamellar structure and the other with a lamellar repeat of 6.1 nm corresponding to bilayers of pure phospholipid.

Structure, Composition, and Peptide Binding Properties of Detergent Soluble Bilayers and Detergent Resistant Rafts

Lipid bilayers composed of unsaturated phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol are thought to contain microdomains that have similar detergent insolubility characteristics as rafts isolated from cell plasma membranes. We chemically characterized the fractions corresponding to detergent soluble membranes (DSMs) and detergent resistant membranes (DRMs) from 1:1:1 PC:SM:cholesterol, compared the binding properties of selected peptides to bilayers with the compositions of DSMs and DRMs, used differential scanning calorimetry to identify phase transitions, and determined the structure of DRMs with x-ray diffraction. Compared with the equimolar starting material, DRMs were enriched in both SM and cholesterol. Both transmembrane and interfacial peptides bound to a greater extent to DSM bilayers than to DRM bilayers, likely because of differences in the mechanical properties of the two bilayers. Thermograms from 1:1:1 PC:SM:cholesterol from 3 to 70°C showed no evidence for a liquid-ordered to liquid-disordered phase transition. Over a wide range of osmotic stresses, each x-ray pattern from equimolar PC:SM:cholesterol or DRMs contained a broad wide-angle band at 4.5 Å, indicating that the bilayers were in a liquid-crystalline phase, and several sharp low-angle reflections that indexed as orders of a single lamellar repeat period. Electron density profiles showed that the total bilayer thickness was 57 Å for DRMs, which was ∼5 Å greater than that of 1:1:1 PC:SM:cholesterol and 10 Å greater than the thickness of bilayers with the composition of DSMs. These x-ray data provide accurate values for the widths of raft and nonraft bilayers that should be important in understanding mechanisms of protein sorting by rafts.

Phase separations of α-tocopherol in aqueous dispersions of distearoylphosphatidylethanolamine

The effect of α-tocopherol on the structure and thermotropic phase behaviour of distearoylphosphatidylethanolamine was examined by using synchrotron X-ray diffraction methods. There was evidence that α-tocopherol does not distribute randomly in the dispersed phospholipid but instead phospholipid phases enriched in α-tocopherol are formed. Heating codispersions from lamellar gel phase induced formation of hexagonal-II phase at temperatures below the main transition of the pure phospholipid and which were enriched in α-tocopherol. Codispersions containing 5 or 10 mol% α-tocopherol were induced to form a cubic phase at temperatures above the lamellar to hexagonal-II phase transition. Such phases were not observed in codispersions containing 2.5 or 20 mol% α-tocopherol in which only lamellar and hexagonal-II phases were formed. The space group of the cubic phases were tentatively assigned as Pn3 m. Equilibration of codispersions at 4 °C results in the formation of lamellar crystalline phases enriched in α-tocopherol and phase separated domains of pure phospholipid. Two lamellar crystalline phases were characterized on the basis of their particular wide-angle X-ray scattering patterns. The lamellar crystalline phases were also distinguished from other lamellar phases of the pure phospholipid by the lamellar repeat. Partitioning of α-tocopherol into phosphatidylethanolamine domains in membranes may introduce instability into the structure.

Crystallization within Melt Ordered Semicrystalline Block Copolymers: Exploring the Coexistence of Microphase-Separated and Spherulitic Morphologies

The crystalline and microphase-separated morphology of semicrystalline block copolymers are compared in bulk (0.5-1 mm) and thin films (5-0 Im), as characterized by transmission electron microscopy (TEM) and polarized light microscopy (PLM). Bulk films of a triblock copolymer of polystyrene, polybutadiene, and polycaprolactone, (PS)0.35(PB)0.15(PCL)0.5, where the subscripts denote the mass fraction, show a lamellar-cylindrical microphase-separated morphology with a lamellar repeat of =62 nm, as observed by TEM. Bulk films of a diblock copolymer consisting of (PB)0.2(PCL)0.8 show an imperfect cylindrical microphase-separated structure by TEM coexisting with crystalline lamellae of PCL. For both bulk specimens, the PLM shows a speckle pattern of birefringence that confirms the PCL is crystallized within the microphase-separated structure, but no large-scale spherulites are observed by PLM. The microstructure in the as-cast thin films (5-10 Im) by TEM is lamellar-cylindrical for the triblock copolymer and cylindrical for the diblock copolymer, as described above for the bulk specimens. Upon thermal treatment of these thin films, large and well-defined PCL spherulites are observed by PLM for both block copolymers. TEM of the thermally treated triblock copolymer thin films shows that, after the formation of spherulites, the microphase structure is no longer lamellar-cylindrical but entirely dominated by PCL lamellar crystals. In contrast, TEM of the thermally treated diblock copolymer thin films shows that the PCL lamellar crystals coexist with the PB microphase-separated cylinders after the formation of PCL spherulites. Bulk specimens could not be induced to form spherulites under similar thermal treatment conditions.

Electrostatically induced undulations of lamellar DNA-lipid complexes

We consider DNA-cationic lipid complexes that form lamellar stacks of lipid bilayers with parallel DNA strands intercalated in between. We calculate the electrostatically induced elastic deformations of the lipid bilayers. It is found that the membranes undulate with a periodicity that is set by the DNA interaxial distance. As a consequence the lamellar repeat distance changes resulting in a swelling or compression of the lamellar stack. Such undulations may be responsible for the intermembrane coupling between DNA strands in different layers as it is observed experimentally.

Structural and morphological investigations of the formation of quasi-crystalline phases of 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG)

1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in 100 mM NaCl at pH 7 forms a quasi-crystalline gel phase (Lc phase) when stored for several days or longer at 4°C. We studied the structure and morphology of this phase by freeze–fracture electron microscopy, synchrotron X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR). The phase behavior was also followed by differential scanning calorimetry. Freeze–fracture electron microscopy revealed two different distinct morphological aggregates for the Lc phase, namely flat multilamellar sheets and cochleate cylinders in an approximately 1:1 ratio. FTIR investigations showed that the hydrocarbon chains are tightly packed within an orthorhombic subcell lattice. The frequency shift of the ester carbonyl bands to lower wavenumbers (1732 cm−1) indicates a direct involvement of the carbonyl groups in hydrogen bonding with hydroxy groups. The freeze–fracture electron microscopic and X-ray investigation reveal very tightly packed lamellar aggregates. The small lamellar repeat distance of 4.75 nm indicates that only a thin water layer is present between the lipid bilayers. The set of reflections observed in the wide angle X-ray region resembles that of the crystalline Lc phase observed in phosphatidylcholines. Upon cooling, the lamellar repeat distance observed in the liquid-crystalline phase increases drastically until the transition into the lamellar gel phase occurs. Then, the reflection in the low angle region becomes broad and diffuse. This finding agrees with the electron microscopic results that upon cooling multilamellar aggregates are destroyed and unilamellar DMPG vesicles are formed below the main phase transition. The unilamellar vesicles fuse to lamellar stacks upon low temperature storage and these then roll up into cochleate cylinders. The observed behavior is attributed to the unique properties of the glycerol headgroup in being able to replace water molecules in the hydration shell.