Tom Dvir

Following the structural changes during zinc-induced crystallization of charged membranes using time-resolved solution X-ray scattering

Zinc ions are highly abundant in biological systems and interact with various enzymes, proteins and biomembranes. In this paper, an in-house state-of-the-art time-resolved solution X-ray scattering setup was used to study the interactions of divalent ions with charged membranes. We show that unlike calcium ions that strongly couple and crystallize charged membranes very rapidly, zinc ions exhibit a fast time scale (seconds) for the strong coupling of the bilayers and a much slower one (hours) for the 2D lateral crystallization of the bilayers. This is attributed to the smaller zinc ion size (compared to calcium ions), which requires higher energy to shed its hydration shells. The rate of crystallization depends on the structure of the lipid tails and is slower for the unsaturated lipid, DOPS, than the saturated lipid, DLPS. We attribute this to the stronger steric repulsion between unsaturated DOPS tails, which have kinks, and to the weaker cohesive electrostatic energy, induced by the zinc ions, due to the larger area per head-group of DOPS. The Avrami model for a 2D growth mechanism with an instantaneous nucleation describes well the crystallization process. The crystallization involves various structural changes in the bilayer structure and lipid conformations within each bilayer. In this paper, we present those structural changes as a function of time.

Effect of temperature on the structure of charged membranes.

Interactions between charged and neutral self-assembled phospholipid membranes are well understood and take into account temperature dependence. Yet, the manner in which the structure of the membrane is affected by temperature was hardly studied. Here we study the effect of temperature on the thickness, area per lipid, and volume per lipid of charged membranes. Two types of membranes were studied: membranes composed of charged lipids and dipolar (neutral) membranes that adsorbed divalent cations and became charged. Small-angle X-ray scattering data demonstrate that the thickness of charged membranes decreases with temperature. Wide-angle X-ray scattering data show that the area per headgroup increases with temperature. Intrinsically charged membranes linearly thin with temperature, whereas neutral membranes that adsorb divalent ions and become charged show an exponential decrease of their thickness. The data indicate that, on average, the tails shorten as the temperature rises. We attribute this behavior to higher lipid tail entropy and to the weaker electrostatic screening of the charged headgroups, by their counterions, at elevated temperatures. The latter effect leads to stronger electrostatic repulsion between the charged headgroups that increases the area per headgroup and decreases the bilayer thickness.

Entropic Attraction Condenses Like-Charged Interfaces Composed of Self-Assembled Molecules

Like-charged solid interfaces repel and separate from one another as much as possible. Charged interfaces composed of self-assembled charged-molecules such as lipids or proteins are ubiquitous. The present study shows that although charged lipid-membranes are sufficiently rigid, in order to swell as much as possible, they deviate markedly from the behavior of typical like-charged solids when diluted below a critical concentration (ca. 15 wt %). Unexpectedly, they swell into lamellar structures with spacing that is up to four times shorter than the layers should assume (if filling the entire available space). This process is reversible with respect to changing the lipid concentration. Additionally, the research shows that, although the repulsion between charged interfaces increases with temperature, like-charged membranes, remarkably, condense with increasing temperature. This effect is also shown to be reversible. Our findings hold for a wide range of conditions including varying membrane charge density, bending rigidity, salt concentration, and conditions of typical living systems. We attribute the limited swelling and condensation of the net repulsive interfaces to their self-assembled character. Unlike solids, membranes can rearrange to gain an effective entropic attraction, which increases with temperature and compensates for the work required for condensing the bilayers. Our findings provide new insight into the thermodynamics and self-organization of like-charged interfaces composed of self-assembled molecules such as charged biomaterials and supramolecular assemblies that are widely found in synthetic and natural constructs.

Effect of temperature on the interactions between dipolar membranes.

It is well-known that phospholipids in aqueous environment self-assemble into lamellar structures with a repeat distance governed by the interactions between them. Yet, the understanding of these interactions is incomplete. In this paper, we study the effect of temperature on the interlamellar interactions between dipolar membranes. Using solution small-angle X-ray scattering (SAXS), we measured the repeat distance between 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) bilayers at different temperatures and osmotic stresses. We found that when no pressure is applied the lamellar repeat distance, D, decreases and then increases with increasing temperature. As the osmotic stress increases, D decreases with temperature and then increases to a limited extent, until at sufficiently high pressure D decreases with temperature in all the examined range. We then reconstructed experimentally the equation of state and fit it with a modified interaction model that takes into account the temperature dependence of the fluctuation term. Finally, we showed how the thickness of DLPC membranes decreases with temperature.

Charged membranes under confinement induced by polymer-, salt-, or ionic liquid solutions

Osmotic pressure confines and decreases the lamellar distance between membranes in a stack and may also change the phase or structure of lipid bilayers. Using solution X-ray scattering, we determined the structure and interactions of saturated (DLPS) and unsaturated (DOPS) charged lipids under osmotic stress, generated by osmolyte solutions consisting of polyethylene glycol (PEG), NaCl, or the ionic liquid EMIES. The measured pressure–distance curves were fit to the theoretical free energy of charged liquid membranes that included numerical solutions of the Poisson–Boltzmann theory, modified to account for nonelectrostatic interactions between counterions and the surface and between lipid molecules on the surface. Charged membranes exhibit a non-ideal swelling behaviour that is consistent with partial melting of the lamellar phase into a coexisting, disordered phase. The disordered phase applies an osmotic stress to the lamellar phase and decreases the spacing between bilayers in the lamellar phase. We showed that in pure water, the pressure of the disordered phase increased with the lipid volume fraction or temperature. Addition of osmolytes reduced the lamellar spacing. The numerical solutions that fit the data showed that at the critical osmolyte concentration, a small, sharp drop in the water spacing between bilayers is expected owing to massive adsorption of the counterions onto the membrane. The adsorption increases with the osmolyte concentration, and is accompanied by a lateral phase separation into domains of neutral (counterion adsorbing) and charged (non-adsorbing) lipid molecules. In the case of the ionic liquid, this transition was followed by a liquid-to-crystal phase transition of the bilayers. With NaCl the transition to the crystal phase required a significantly higher osmolyte concentration. In the crystal phase that was induced by the ionic liquid, the lipid molecules were more tilted than in the crystal phase formed by NaCl. A further increase of the ionic liquid concentration melted the lipid crystal phase into the gel phase.

Charging and softening, collapse, and crystallization of dipolar phospholipid membranes by aqueous ionic liquid solutions.

Ionic liquids have a variety of unique controllable structures and properties. These properties may be used to tailor the self-assembly of charged and dipolar biomolecules. Using solution X-ray scattering, we measured the structure of Dilauryl(C12:0)-sn-glycero-3-phospho-l-choline (DLPC), a dipolar (or zwitterionic) lipid, in the water-soluble room temperature ionic liquid Ethyl Methyl Imidazolium Ethyl Sulfate (EMIES) and mixtures of EMIES and water. We find that the interaction between the lipid bilayers is dominated by the balance between the charging of the polar headgroups by the ionic liquid, softening of the bilayer, and the osmotic pressure induced by the solvent. This balance leads to the following changes with increasing ionic liquid concentration: an incomplete unbinding transition from an attractive regime to a swollen regime of the lamellar phase formed by the bilayers. The swollen phase is followed by a collapse of the bilayers into a highly desolvated lamellar phase at some critical EMIES concentration, and eventually formation of lipid-crystalline phase, at very high EMIES concentrations. The latter phase is revealed by wide-angle X-ray scattering (WAXS) from the lipid solutions, showing multiple Bragg peaks, consistent with highly ordered structures. These structures were not observed in any other type of aqueous solutions containing monovalent or multivalent ions. The kinetics and temperature dependence of these transitions were also determined.

Osmotic Stress Induced Desorption of Calcium Ions from Dipolar Lipid Membranes

The interaction between multivalent ions and lipid membranes with saturated tails and dipolar (net neutral) headgroups can lead to adsorption of the ions onto the membrane. The ions charge the membranes and contribute to electrostatic repulsion between them, in a similar manner to membranes containing charged lipids. Using solution X-ray scattering and the osmotic stress method, we measured and modeled the pressure-distance curves between partially charged membranes containing mixtures of charged (1,2-dilauroyl-sn-glycero-3-phospho-l-serine, DLPS) and dipolar (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC) lipids over a wide range of membrane charge densities. We then compared these pressure-distance curves with those of DLPC membranes in the presence of 10 mM CaCl2. Our data and modeling show that when low osmotic stress is applied to the DLPC bilayers, the membrane charge density is equivalent to that of a charged membrane containing ca. 4 mol % DLPS and 96 mol % DLPC. As the osmotic stress increased, the charge density of the DLPC membrane decreased and resembled that of a membrane containing ca. 1 mol % DLPS. These data are consistent with desorption of the calcium ions from the DLPC membrane with increasing osmotic stress.

High-density carriers at a strongly coupled interface between graphene and a three-dimensional topological insulator

We report on a strongly coupled bilayer graphene (BLG) Bi2Se3 device with a junction resistance of less than 1.5 kΩμm. This device exhibits unique behavior at the interface, which cannot be attributed to either material in absence of the other. We observe quantum oscillations in the magnetoresistance of the junction, indicating the presence of well-resolved Landau levels due to hole carriers of unknown origin with a very large Fermi surface. These carriers, found only at the interface, could conceivably arise due to significant hole doping of the bilayer graphene with charge transfer on the order of 2×10 cm−2, or due to twist angle dependent mini-band transport.

Structure and Intermolecular Interactions between L-Type Straight Flagellar Filaments

Bacterial mobility is powered by rotation of helical flagellar filaments driven by rotary motors. Flagellin isolated from the Salmonella Typhimurium SJW1660 strain, which differs by a point mutation from the wild-type strain, assembles into straight filaments in which flagellin monomers are arranged in a left-handed helix. Using small-angle x-ray scattering and osmotic stress methods, we investigated the structure of SJW1660 flagellar filaments as well as the intermolecular forces that govern their assembly into dense hexagonal bundles. The scattering data were fitted to models, which took into account the atomic structure of the flagellin subunits. The analysis revealed the exact helical arrangement and the super-helical twist of the flagellin subunits within the filaments. Under osmotic stress, the filaments formed two-dimensional hexagonal bundles. Monte Carlo simulations and continuum theories were used to analyze the scattering data from hexagonal arrays, revealing how the bundle bulk modulus and the deflection length of filaments in the bundles depend on the applied osmotic stress. Scattering data from aligned flagellar bundles confirmed the theoretically predicated structure-factor scattering peak line shape. Quantitative analysis of the measured equation of state of the bundles revealed the contributions of electrostatic, hydration, and elastic interactions to the intermolecular forces associated with bundling of straight semi-flexible flagellar filaments.