Does sucrose influence the properties of DMPC vesicles?

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

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Structural characterization of the micelle-vesicle transition in lecithin-bile salt solutions.

Small-angle neutron scattering (SANS) and dynamic light scattering (QLS) are used to characterize the aggregates found upon dilution of mixed lecithin-bile salt micelles. Molar ratios of lecithin (L) to taurocholate (TC) studied varied from 0.1 to 1, and one series contained cholesterol (Ch). Mixed aggregates of L and taurodeoxycholate (TDC) at ratios of 0.4 and 1 were also examined. In all cases the micelles are cylindrical or globular and elongate upon dilution. The radius of the mixed micelles varies only slightly with the overall composition of lecithin and bile salt which indicates that the composition of the cylindrical micelle body is nearly constant. The transition from micelles to vesicles is a smooth transformation involving a region where micelles and vesicles coexist. SANS measurements are more sensitive to the presence of two aggregate populations than QLS. Beyond the coexistence region the vesicle size and degree of polydispersity decrease with dilution. Incorporation of a small amount of cholesterol in the lipid mixture does not affect the sequence of observed aggregate structures.     

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

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

Osmotic swelling of unilamellar vesicles by the stopped-flow light scattering method. Elastic properties of vesicles

Unilamellar vesicles of l-α-dimyristoylphophatidylcholine have been prepared by the ether injection technique. Gel filtration on Sephacryl S1000 was used to obtain fractions of narrow polydispersity, of radius from 300 to 600 Å. Dynamic light scattering was used to determine the change in size of these vesicles in response to an osmotic pressure drop, and its dependence on vesicle size. The amplitude of swelling (ΔR/R) is linearly proportional to the osmotic pressure difference across the bilayer. We have determined the elastic area stretching modulus using a theory of membrane elasticity: it depends on the vesicle radius in the range of size studied. Vesicles having radius smaller than 400 Å show little or no swelling.     

Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparison with small sonicated vesicles

Dioctadecyldimethylammonium chloride (DODAC) unilamellar liposomes with a mean external diameter of 0.5 μm and sharp gel-to-liquid-crystalline phase transition temperatures (T_c) were obtained by chloroform vaporization and compared with small sonicated DODAC vesicles. Sucrose, impermeant through large DODAC liposomes and sonicated vesicles, was used for internal volume determinations. The internal volumes for large DODAC liposomes and sonicated DODAC vesicles were 9.0 ± 1.3 and 0.13 ± 0.2 l/mol, respectively. Ideal osmometer behaviour, towards KCl (0–50 mM) and sucrose, was observed only for large DODAC liposomes. Sonicated DODAC vesicles were osmotically non-responsive towards sucrose and flocculated upon addition of KCl. At temperatures near the T_c, a steep increase in the initial shrinkage rate and a minimum for the total extent of shrinkage were observed for large DODAC liposomes. Large DODAC liposomes are proposed as an adequate synthetic membrane model.     

Liver glycogen in type 2 diabetic mice is randomly branched as enlarged aggregates with blunted glucose release

Glycogen is a vital highly branched polymer of glucose that is essential for blood glucose homeostasis. In this article, the structure of liver glycogen from mice is investigated with respect to size distributions, degradation kinetics, and branching structure, complemented by a comparison of normal and diabetic liver glycogen. This is done to screen for differences that may result from disease. Glycogen α-particle (diameter ～ 150 nm) and β-particle (diameter ～ 25 nm) size distributions are reported, along with in vitro γ-amylase degradation experiments, and a small angle X-ray scattering analysis of mouse β-particles. Type 2 diabetic liver glycogen upon extraction was found to be present as large loosely bound, aggregates, not present in normal livers. Liver glycogen was found to aggregate in vitro over a period of 20 h, and particle size is shown to be related to rate of glucose release, allowing a structure-function relationship to be inferred for the tissue specific distribution of particle types. Application of branching theories to small angle X-ray scattering data for mouse β-particles revealed these particles to be randomly branched polymers, not fractal polymers. Together, this article shows that type 2 diabetic liver glycogen is present as large aggregates in mice, which may contribute to the inflexibility of interconversion between glucose and glycogen in type 2 diabetes, and further that glycogen particles are randomly branched with a size that is related to the rate of glucose release.     

Lipid-sugar interactions : relevance to anhydrous biology

The ability of seeds and other anhydrous plant forms to survive the withdrawal of water must involve a mechanism for protecting the integrity of cellular membranes. Evidence from animal systems implicates sugars as protective components, and we have tested the changes in mesomorphic phase state of phospholipid model membranes upon hydration and dehydration in the presence of sucrose and/or sucrose plus raffinose. X-ray diffraction studies of dry dimyristoylphosphatidylcholine (DMPC) indicate that the presence of sucrose lowers the chain order/disorder transition temperature to that of hydrated lipid; likewise, the lamellar repeat spacings showed the dry DMPC/sucrose mixture to be similar to that of the hydrated lipid. These results support the proposed potential of sugars to substitute for water in biomembranes. If sucrose is to serve as a protectant during desiccation of seeds, its tendency to crystallize would lessen its effectiveness. Raffinose is known to serve as an inhibitor of sucrose crystallization, and is abundant in seeds. The addition of raffinose to make DMPC/sucrose/raffinose mixtures (1/1/0.3 mass ratio) prevented sucrose crystallization, suggesting this as a possible in vivo role for raffinose.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Structure of clathrin-coated vesicles from small-angle scattering experiments

Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D20 solvent concentrations. The model used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. Å constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein shell (clathrin) and a central protein shell (accessory polypeptides and receptors) of approximately six times higher density. For the X-ray scattering and neutron contrast variation data, the polydispersity of the samples is of the order of 90 Å (full-width-at-half-maximum value) and the average outer radius is approximately 400 Å. The inner high-density shell has inner and outer radii of 115 and 190 Å, respectively. Å simultaneous fit to the three neutron contrast variation data sets identifies the lipid membrane with a thickness of 40 Å and an outer radius of 196 Å. Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 × 106 Da. The coated vesicles consist, on average, of approximately 85 % protein and 15 lipids. About 40% of the protein mass is situated in the central high-density shell, which gives a large amount of protein in the lipid membrane. The densities of the central shell and the lipid membrane show that the hydration is small in the central region. Å comparison of the total mass, the mass distribution, and the structure of the average-size particles with the barrel structure shows that the accessory polypeptides are incorporated in the lipid membrane. The results from the neutron data for the reassembled coats show that the structure of these particles is very similar to the structure of the native coats. The main difference is a higher density of the central protein shell, which shows that the membrane is replaced by protein in the reassembled coats.     

Complex biomembrane mimetics on the sub-nanometer scale

Biomimetic lipid vesicles are indispensable tools for gaining insight into the biophysics of cell physiology on the molecular level. The level of complexity of these model systems has steadily increased, and now spans from domain-forming lipid mixtures to asymmetric lipid bilayers. Here, we review recent progress in the development and application of elastic neutron and X-ray scattering techniques for studying these systems in situ and under physiologically relevant conditions on the nanometer to sub-nanometer length scales. In particular, we focus on: (1) structural details of coexisting liquid-ordered and liquid-disordered domains, including their thickness and lipid packing mismatch as a function of a size transition from nanoscopic to macroscopic domains; (2) membrane-mediated protein partitioning into lipid domains; (3) the role of the aqueous medium in tuning interactions between membranes and domains; and (4) leaflet-specific structure in asymmetric bilayers and passive lipid flip-flop.     

Pore formation kinetics in membranes, determined from the release of marker molecules out of liposomes or cells

We discuss the efflux of entrapped marker material from liposomes or cells through pores in the membrane, being monitored by the time course of a certain signal F (e.g., fluorescence emission). This is expressed in terms of an appropriate normalized function of time, the so-called efflux function E(t). Under conditions frequently encountered in practice the measured E(t) can be easily related to the forward rate of pore formation if the liposomes/cells are monodisperse in size. In the basic case of a time-independent rate law it turns out that E(t) must be single exponential. Deviations from such a simple functional behavior might be due to a fairly broad distribution of liposome/cell sizes and/or a more complicated pore formation mechanism. A relevant evaluation of original data is demonstrated making use of experimental results obtained with small unilamellar lipid vesicles where pores are induced by the antibiotic peptide alamethicin. This includes the application of a general method to eliminate the effect of a given liposome/cell size distribution.     

Vesicle Adhesion and Fusion Studied by Small-Angle X-Ray Scattering

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca²⁺ or Mg²⁺ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ～1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering.     

Apolipophorin III interaction with model membranes composed of phosphatidylcholine and sphingomyelin using differential scanning calorimetry

Apolipophorin III (apoLp-III) from Locusta migratoria was employed as a model apolipoprotein to gain insight into binding interactions with lipid vesicles. Differential scanning calorimetry (DSC) was used to measure the binding interaction of apoLp-III with liposomes composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SM). Association of apoLp-III with multilamellar liposomes occurred over a temperature range around the liquid crystalline phase transition (L_α). Qualitative and quantitative data were obtained from changes in the lipid phase transition upon addition of apoLp-III. Eleven ratios of DMPC and SM were tested from pure DMPC to pure SM. Broadness of the phase transition (T_1/2), melting temperature of the phase transition (T_m) and enthalpy were used to determine the relative binding affinity to the liposomes. Multilamellar vesicles composed of 40% DMPC and 60% SM showed the greatest interaction with apoLp-III, indicated by large T_1/2 values. Pure DMPC showed the weakest interaction and liposomes with lower percentage of DMPC retained domains of pure DMPC, even upon apoLp-III binding indicating demixing of liposome lipids. Addition of apoLp-III to rehydrated liposomes was compared to codissolved trials, in which lipids were rehydrated in the presence of protein, forcing the protein to interact with the lipid system. Similar trends between the codissolved and non-codissolved trials were observed, indicating a similar binding affinity except for pure DMPC. These results suggested that surface defects due to non-ideal packing that occur at the phase transition temperature of the lipid mixtures are responsible for apolipoprotein-lipid interaction in DMPC/SM liposomes.     

Shape and size of bovine rhodopsin: A small-angle X-ray scattering study of a rhodopsin-detergent complex

Rhodopsin is extracted from rod outer segments of retinas with dodecyldimethylamine oxide (DDAO), a non-ionie detergent. The rhodopsin-DDAO complex is characterized by binding experiments, gel filtration, sedimentation, densimetry; its homogeneity, chemical composition, weight and partial specific volume are determined. The complex turns out to be a reasonably monodisperse association of one rhodopsin and 156 DDAO molecules. The rhodopsin-DDAO complex and the detergent micelles are studied by small-angle X-ray scattering techniques using a water/sucrose solvent of variable density. The experiments are performed on an absolute scale; mainly the value and curvature of the scattering curves at zero angle are exploited. The structure of the complex and of the micelles is shown to be independent of sucrose. Under these conditions the final result of the X-ray scattering study of each type of particle is the numerical value of a set of five parameters: molecular weight, volume and radius of gyration of the volume occupied by the particles, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of rhodopsin and of the detergent moiety are very near to each other. The analysis of these parameters leads to the determination of the size and shape of the detergent micelles and to an estimate of the size and shape of the volumes occupied by protein and by detergent in the complex. We find rhodopsin to be a very elongated molecule (maximum diameter ~95 Å) which spans a flat detergent micelle. These results suggest that in the rod outer segment discs the rhodopsin molecules span the membranes, that the rhodopsin molecules of the two opposite membranes of each disc come near to each other and that a high fraction of the intra-disc space is occupied by rhodopsin.     

Contrast variation studies of clathrin coated vesicles by small-angle neutron scattering

Structural information on clathrin coated vesicles has been obtained by small angle neutron scattering using contrast variation. A characteristic peak in the neutron scattering profile, which is apparent in 75 % D₂O, as well as in H₂O, disappears when contrast matching the protein component of the coated vesicles in 42% D₂O. Neutron, as well as dynamic, light scattering give a coated vesicle size of about 900 Å in H₂O and D₂O, but for neutron scattering the diameter decreases when matching out the protein coat of the clathrin coated vesicles. From the match point for the clathrin coated vesicles it is demonstrated that the clathrin cages do contain internal membrane. The mass of 34 MD and composition of 75% protein and 25% lipid found from the analysis of the small-angle scattering data are both in good agreement with the values reported in the literature. Electron microscopy gives an average outer diameter of 880 Å for the coated vesicles and an average diameter of 460 Å for the vesicle itself. Offprint requests to: Correspondence to: R. Bauer     

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.     

Kinetics of the lamellar gel–fluid transition in phosphatidylcholine membranes in the presence of sugars

Phase diagrams are presented for dipalmitoylphosphatidylcholine (DPPC) in the presence of sugars (sucrose) over a wide range of relative humidities (RHs). The phase information presented here, determined by small angle X-ray scattering (SAXS), is shown to be consistent with previous results achieved by differential scanning calorimetry (DSC). Both techniques show a significant effect of sucrose concentration on the phase behaviour of this phospholipid bilayer. An experimental investigation into the effect of sugars on the kinetic behaviour of the gel to fluid transition is also presented showing that increasing the sugar content appears to slightly increase the rate at which the transition occurs.     

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.