Calcium Triggered Lα-H2 Phase Transition Monitored by Combined Rapid Mixing and Time-Resolved Synchrotron SAXS

BACKGROUND: Awad et al. reported on the Ca(2+)-induced transitions of dioleoyl-phosphatidylglycerol (DOPG)/monoolein (MO) vesicles to bicontinuous cubic phases at equilibrium conditions. In the present study, the combination of rapid mixing and time-resolved synchrotron small-angle X-ray scattering (SAXS) was applied for the in-situ investigations of fast structural transitions of diluted DOPG/MO vesicles into well-ordered nanostructures by the addition of low concentrated Ca(2+) solutions. METHODOLOGY/PRINCIPAL FINDINGS: Under static conditions and the in absence of the divalent cations, the DOPG/MO system forms large vesicles composed of weakly correlated bilayers with a d-spacing of approximately 140 A (L(alpha)-phase). The utilization of a stopped-flow apparatus allowed mixing these DOPG/MO vesicles with a solution of Ca(2+) ions within 10 milliseconds (ms). In such a way the dynamics of negatively charged PG to divalent cation interactions, and the kinetics of the induced structural transitions were studied. Ca(2+) ions have a very strong impact on the lipidic nanostructures. Intriguingly, already at low salt concentrations (DOPG/Ca(2+)>2), Ca(2+) ions trigger the transformation from bilayers to monolayer nanotubes (inverted hexagonal phase, H(2)). Our results reveal that a binding ratio of 1 Ca(2+) per 8 DOPG is sufficient for the formation of the H(2) phase. At 50 degrees C a direct transition from the vesicles to the H(2) phase was observed, whereas at ambient temperature (20 degrees C) a short lived intermediate phase (possibly the cubic Pn3m phase) coexisting with the H(2) phase was detected. CONCLUSIONS/SIGNIFICANCE: The strong binding of the divalent cations to the negatively charged DOPG molecules enhances the negative spontaneous curvature of the monolayers and causes a rapid collapsing of the vesicles. The rapid loss of the bilayer stability and the reorganization of the lipid molecules within ms support the argument that the transition mechanism is based on a leaky fusion of the vesicles

The ELBA Force Field for Coarse-Grain Modeling of Lipid Membranes

A new coarse-grain model for molecular dynamics simulation of lipid membranes is presented. Following a simple and conventional approach, lipid molecules are modeled by spherical sites, each representing a group of several atoms. In contrast to common coarse-grain methods, two original (interdependent) features are here adopted. First, the main electrostatics are modeled explicitly by charges and dipoles, which interact realistically through a relative dielectric constant of unity (). Second, water molecules are represented individually through a new parametrization of the simple Stockmayer potential for polar fluids; each water molecule is therefore described by a single spherical site embedded with a point dipole. The force field is shown to accurately reproduce the main physical properties of single-species phospholipid bilayers comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) in the liquid crystal phase, as well as distearoylphosphatidylcholine (DSPC) in the liquid crystal and gel phases. Insights are presented into fundamental properties and phenomena that can be difficult or impossible to study with alternative computational or experimental methods. For example, we investigate the internal pressure distribution, dipole potential, lipid diffusion, and spontaneous self-assembly. Simulations lasting up to 1.5 microseconds were conducted for systems of different sizes (128, 512 and 1058 lipids); this also allowed us to identify size-dependent artifacts that are expected to affect membrane simulations in general. Future extensions and applications are discussed, particularly in relation to the methodology's inherent multiscale capabilities

The Effects of Gramicidin on the Structure of Phospholipid Assemblies

Gramicidin is an antibiotic peptide that can be incorporated into the monolayers of cell membranes. Dimerization through hydrogen bonding between gramicidin monomers in opposing leaflets of the membrane results in the formation of an iontophoretic channel. Surrounding phospholipids influence the gating properties of this channel. Conversely, gramicidin incorporation has been shown to affect the structure of spontaneously formed lipid assemblies. Using small-angle x-ray diffraction and model systems composed of phospholipids and gramicidin, the effects produced by gramicidin on lipid layers were measured. These measurements explore how peptides are able to modulate the spontaneous curvature properties of phospholipid assemblies. The reverse hexagonal, H(II), phase formed by dioleoylphosphatidylethanolamine (DOPE) monolayers decreased in lattice dimension with increasing incorporation of gramicidin. This indicated that gramicidin itself was adding negative curvature to the lipid layers. In this system, gramicidin was measured to have an apparent intrinsic radius of curvature, [Formula: see text] of −7.1 Å. The addition of up to 4 mol% gramicidin in DOPE did not result in the monolayers becoming stiffer, as measured by the monolayer bending moduli. Dioleoylphosphatidylcholine (DOPC) alone forms the lamellar (L(α)) phase when hydrated, but undergoes a transition into the reverse hexagonal (H(II)) phase when mixed with gramicidin. The lattice dimension decreases systematically with increased gramicidin content. Again, this indicated that gramicidin was adding negative curvature to the lipid monolayers but the mixture behaved structurally much less consistently than DOPE/gramicidin. Only at 12 mol% gramicidin in dioleoylphosphatidylcholine could an apparent radius of intrinsic curvature of gramicidin [Formula: see text] be estimated as −7.4 Å. This mixture formed monolayers that were very resistant to bending, with a measured bending modulus of 115 kT

Automated Transmission-Mode Scanning Electron Microscopy (tSEM) for Large Volume Analysis at Nanoscale Resolution

Transmission-mode scanning electron microscopy (tSEM) on a field emission SEM platform was developed for efficient and cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizing the resolution and dynamic range necessary for discriminating key subcellular structures, such as small axonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomes which are critical for neuronal function. Individual image fields from the tSEM system were up to 4,295 µm2 (65.54 µm per side) at 2 nm pixel size, contrasting with image fields from a modern transmission electron microscope (TEM) system, which were only 66.59 µm2 (8.160 µm per side) at the same pixel size. The tSEM produced outstanding images and had reduced distortion and drift relative to TEM. Automated stage and scan control in tSEM easily provided unattended serial section imaging and montaging. Lens and scan properties on both TEM and SEM platforms revealed no significant nonlinear distortions within a central field of ~100 µm2 and produced near-perfect image registration across serial sections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCT™ or Fiji/TrakEM2 software. Axial resolution limits the analysis of small structures contained within a section (~45 nm). Since this new tSEM is non-destructive, objects within a section can be explored at finer axial resolution in TEM tomography with current methods. Future development of tSEM tomography promises thinner axial resolution producing nearly isotropic voxels and should provide within-section analyses of structures without changing platforms. Brain was the test system given our interest in synaptic connectivity and plasticity; however, the new tSEM system is readily applicable to other biological systems.This study was funded by United States National Institutes of Health (http://www.nih.gov; grant numbers NS021184, NS074644, and MH095980 to KMH) and Texas Emerging Technologies Fund (http://governor.state.tx.us/ecodev/etf/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

Differential Effects of the Hydrophobic Surfactant Proteins on the Formation of Inverse Bicontinuous Cubic Phases

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce formation of the bicontinuous structures and faster adsorption by similar mechanisms, then they should also have differential ability to form the cubic phases. To test this hypothesis, we measured small angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the doserelated ability of the combined proteins to induce the cubic phases at temperatures more than 25°C below the point at which POPE alone forms the curved inverse-hexagonal phase. With SP-C, diffraction from cubic structures was either absent or present only with larger amounts of protein at low intensities. The correlation between the structural effects of inducing curved structures and the functional effects on the rate of adsorption fits with the model in which SP-B promotes adsorption by facilitating formation of a negatively curved, rate-limiting intermediate structure

Combination of the anti-tumour cell ether lipid edelfosine with sterols abolishes haemolytic side effects of the drug

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is an anti-tumour cell ether lipid with surface-active properties. Pure edelfosine can be dispersed in aqueous media in the form of micelles. One important, negative side effect of edelfosine is that it is highly haemolytic. In this paper, we show that edelfosine can be co-dispersed in water with certain lipids (particularly cholesterol, campesterol or β-sitosterol) so that it gives rise to liposomes. Surface pressure measurements demonstrate that edelfosine is slowly released from these liposomes. In liposomal form, edelfosine remains apoptogenic for a variety of leukemia cell lines, while its haemolytic effect is abolished. The phenomenon is explained on the basis of the complementarity of the molecular geometries of sterols and edelfosine