K. Kjaer

Influence of ether linkages on the structure of double-chain phospholipid monolayers

Abstract The structure of phosphatidylcholine monolayers has been studied by Synchrotron X-ray diffraction at the air/water interface varying the ester and ether linkages of the aliphatic tails at the glycerol backbone. All systems investigated exhibit an oblique lattice structure with extremely large tilt angles of the chains from vertical, even at high lateral pressures. Although no large difference is seen in the isotherms, the replacement of ester by ether linkages causes a reduction of the tilt angle and of the area per molecule. These changes also depend on the position of the ether group with respect to the glycerol backbone and can be understood within a model where the carbonyl group of the ester at the C2 position pulls the attached chain towards the water subphase.

Phases of phosphatidyl ethanolamine monolayers studied by synchrotron x-ray scattering

For the first time, phospholid monolayers at the air/water interface have been studied by x-ray diffraction and reflection all along the isotherm from the laterally isotropic fluid (the so-called LE phase) to the ordered phases. The model used to analyze the data, and the accuracy of the parameters deduced, were tested by comparing the results obtained with two lipids having the same head group but different chain lengths. Compression of the fluid phase leads predominantly to a change of thickness of the hydrophobic moiety, much less of its density, with the head group extension remaining constant. The main transition involves a considerable increase (approximately 10%) of the electron density in the hydrophobic region, a dehydration of the head group and a positional ordering of the aliphatic tails, albeit with low coherence lengths (approximately 10 spacings). On further compression of the film, the ordered phase undergoes a continuous transition. This is characterized by an increase in positional ordering, a discontinuous decrease in lateral compressibility, a decrease in chain tilt angle with respect to the surface normal towards zero and probably also a head group dehydration and ordering.

Lipid membrane templates the ordering and induces the fibrillogenesis of Alzheimer's disease amyloid-β peptide

The lipid membrane has been shown to mediate the fibrillogenesis and toxicity of Alzheimers disease (AD) amyloid‐β (Aβ) peptide. Electrostatic interactions between Aβ40 and the phospholipid headgroup have been found to control the association and insertion of monomeric Aβ into lipid monolayers, where Aβ exhibited enhanced interactions with charged lipids compared with zwitterionic lipids. To elucidate the molecular‐scale structural details of Aβ‐membrane association, we have used complementary X‐ray and neutron scattering techniques (grazing‐incidence X‐ray diffraction, X‐ray reflectivity, and neutron reflectivity) in this study to investigate in situ the association of Aβ with lipid monolayers composed of either the anionic lipid 1,2‐dipalmitoyl‐sn‐glycero‐3‐[phospho‐rac‐(1‐glycerol)] (DPPG), the zwitterionic lipid 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC), or the cationic lipid 1,2‐dipalmitoyl 3‐trimethylammonium propane (DPTAP) at the air‐buffer interface. We found that the anionic lipid DPPG uniquely induced crystalline ordering of Aβ at the membrane surface that closely mimicked the β‐sheet structure in fibrils, revealing an intriguing templated ordering effect of DPPG on Aβ. Furthermore, incubating Aβ with lipid vesicles containing the anionic lipid 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐[phospho‐rac‐(1‐glycerol)] (POPG) induced the formation of amyloid fibrils, confirming that the templated ordering of Aβ at the membrane surface seeded fibril formation. This study provides a detailed molecular‐scale characterization of the early structural fluctuation and assembly events that may trigger the misfolding and aggregation of Aβ in vivo. Our results implicate that the adsorption of Aβ to anionic lipids, which could become exposed to the outer membrane leaflet by cell injury, may serve as an in vivo mechanism of templated‐aggregation and drive the pathogenesis of AD. Proteins 2008.

Crystallinity of the Double Layer of Cadmium Arachidate Films at the Water Surface

A crystalline counterionic layer at the interface between an electrolyte solution and a charged layer of insoluble amphiphilic molecules was observed with grazing incidence synchrotron x-ray diffraction. Uncompressed arachidic films spread over 10–3 molar cadmium chloride solution (pH 8.8) spontaneously form crystalline clusters with coherence lengths of ∼1000 angstroms at 9�C. Ten distinct diffraction peaks were observed, seven of which were attributed to scattering only from a crystalline Cd2+ layer and the other three to scattering primarily from the arachidate layer. The reflections from the Cd2+ layer were indexed according to a 2 x 3 supercell of the arachidate lattice with three Cd2+ ions per cadmium unit cell.

Packing of Ganglioside-Phospholipid Monolayers: An X-Ray Diffraction and Reflectivity Study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structure of mixed ganglioside-phospholipid monolayers was investigated at the air-water interface. Mixed monolayers of 0, 5, 10, 20, and 100 mol% ganglioside GM(1) and the phospholipid dipalmitoylphosphatidylethanolamine (DPPE) were studied in the solid phase at 23 degrees C and a surface pressure of 45 mN/m. At these concentrations and conditions the two components do not phase-separate and no evidence for domain formation was observed. X-ray scattering measurements reveal that GM(1) is accommodated within the host DPPE monolayer and does not distort the hexagonal in-plane unit cell or out-of-plane two-dimensional (2-D) packing compared with a pure DPPE monolayer. The oligosaccharide headgroups were found to extend normally from the monolayer surface, and the incorporation of these glycolipids into DPPE monolayers did not affect hydrocarbon tail packing (fluidization or condensation of the hydrocarbon region). This is in contrast to previous investigations of lipopolymer-lipid mixtures, where the packing structure of phospholipid monolayers was greatly altered by the inclusion of lipids bearing hydrophilic polymer groups. Indeed, the lack of packing disruptions by the oligosaccharide groups indicates that protein-GM(1) interactions, including binding, insertion, chain fluidization, and domain formation (lipid rafts), can be studied in 2-D monolayers using scattering techniques.

Synchrotron X-Ray Study of Lung Surfactant-Specific Protein SP-B in Lipid Monolayers

This work reports the first x-ray scattering measurements to determine the effects of SP-B(1-25), the N-terminus peptide of lung surfactant-specific protein SP-B, on the structure of palmitic acid (PA) monolayers. In-plane diffraction shows that the peptide fluidizes a portion of the monolayer but does not affect the packing of the residual ordered phase. This implies that the peptide resides in the disordered phase, and that the ordered phase is essentially pure lipid, in agreement with fluorescence microscopy studies. X-ray reflectivity shows that the peptide is oriented in the lipid monolayer at an angle of approximately 56 degrees relative to the interface normal, with one end protruding past the hydrophilic region into the fluid subphase and the other end embedded in the hydrophobic region of the monolayer. The quantitative insights afforded by this study lead to a better understanding of the lipid/protein interactions found in lung surfactant systems.

Condensing and fluidizing effects of ganglioside GM1 on phospholipid films

Mixed monolayers of the ganglioside G(M1) and the lipid dipalmitoylphosphatidlycholine (DPPC) at air-water and solid-air interfaces were investigated using various biophysical techniques to ascertain the location and phase behavior of the ganglioside molecules in a mixed membrane. The effects induced by G(M1) on the mean molecular area of the binary mixtures and the phase behavior of DPPC were followed for G(M1) concentrations ranging from 5 to 70 mol %. Surface pressure isotherms and fluorescence microscopy imaging of domain formation indicate that at low concentrations of G(M1) (<25 mol %), the monolayer becomes continually more condensed than DPPC upon further addition of ganglioside. At higher G(M1) concentrations (>25 mol %), the mixed monolayer becomes more expanded or fluid-like. After deposition onto a solid substrate, atomic force microscopy imaging of these lipid monolayers showed that G(M1) and DPPC pack cooperatively in the condensed phase domain to form geometrically packed complexes that are more ordered than either individual component as evidenced by a more extended total height of the complex arising from a well-packed hydrocarbon tail region. Grazing incidence x-ray diffraction on the DPPC/G(M1) binary mixture provides evidence that ordering can emerge when two otherwise fluid components are mixed together. The addition of G(M1) to DPPC gives rise to a unit cell that differs from that of a pure DPPC monolayer. To determine the region of the G(M1) molecule that interacts with the DPPC molecule and causes condensation and subsequent expansion of the monolayer, surface pressure isotherms were obtained with molecules modeling the backbone or headgroup portions of the G(M1) molecule. The observed concentration-dependent condensing and fluidizing effects are specific to the rigid, sugar headgroup portion of the G(M1) molecule.

Amyloid-β Fibrillogenesis Seeded by Interface-Induced Peptide Misfolding and Self-Assembly

The amphipathicity of the natively unstructured amyloid-beta (Abeta40) peptide may play an important role in its aggregation into beta-sheet rich fibrils, which is linked to the pathogenesis of Alzheimers disease. Using the air/subphase interface as a model interface, we characterized Abetas surface activity and its conformation, assembly, and morphology at the interface. Abeta readily adsorbed to the air/subphase interface to form a 20 A thick film and showed a critical micelle concentration of approximately 120 nM. Abeta adsorbed at the air/subphase exhibited in-plane ordering that gave rise to Bragg peaks in grazing-incidence x-ray diffraction measurements. Analysis of the peaks showed that the air/subphase interface induced Abeta to fold into a beta-sheet conformation and to self-assemble into approximately 100 A-sized ordered clusters. The formation of these clusters at the air/subphase interface was not affected by pH, salts, or the presence of sucrose or urea, which are known to stabilize or denature native proteins, suggesting that interface-driven Abeta misfolding and assembly are strongly favored. Furthermore, Abeta at the interface seeded the growth of fibrils in the bulk with a distinct morphology compared to those formed by homogeneous nucleation. Our results indicate that interface-induced Abeta misfolding may serve as a heterogeneous, nucleation-controlled aggregation mechanism for Abeta fibrillogenesis in vivo.

Structure of Cholesterol/Lipid Ordered Domains in Monolayers and Single Hydrated Bilayers

Cell membranes are currently thought to consist of different lipid phases and domains at the nanometer scale. These domains (also known as “lipid rafts”) differ in composition, structure, and stability and consequently in properties and function. They selectively incorporate or exclude specific proteins, and thereby fulfill an important function in cell activity and signaling. The compositions of the different membranes in the cell organelles differ greatly according to their function. Membrane compositions also vary substantially among different types of cells and among organelles within the same cell. To date, thousands of different lipid molecules have been found to exist in the cell membranes, often differing one from the other only in chain length or other parameters not associated with functional-group interaction. The possible advantage of this multiplicity and redundancy may be explained at least in part by the different interactions between the lipid chains, which in turn regulate the composition, organization, and functional parameters of the membrane. Understanding the rules that govern membrane structure is thus crucial to understanding cell biology. Grazing-incidence X-ray diffraction (GIXD) can provide direct information on the organization of laterally periodically ordered lipid sheets. GIXD has been used for studying the structure of two-dimensional (2D) lipid monolayers at the air/water interface. Membranes are, however, bilayers composed of two opposing juxtaposed monolayers, which interact with each other at the hydrophobic side. The structure of the bilayer may thus be conceivably different from that of the corresponding monolayer. To guarantee preservation of the structural integrity of a membrane bilayer with hydrophobic interior and hydrophilic external surfaces, wetting on both sides of the bilayer is required. However, because of the strong X-ray background scattering contribution of liquid water, until now GIXD experiments have been reported on lipid films that were dry at the side of the impinging X-ray beam, or on stacks of bilayers. Such studies should be compared with those of individual hydrated lipid bilayers. This was recently made possible by working with highenergy beams, for which the attenuation resulting from photoelectric absorption by water is weaker, thus preserving more of the X-ray intensity even when the sample is immersed in water. Such a pioneering experiment was performed recently by Miller et al. , who obtained a diffraction signal from a single 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) bilayer. The bilayer was directly deposited on a silicon wafer, thus limiting the diffusion kinetics of the molecules in contact with the wafer. The intensity of the signal was strongly attenuated by the presence of water, which, additionally, gave rise to strong background scattering. Therefore, this method might not succeed in measuring samples with a low amount of crystalline bilayer material, as in this study. Herein, we report the crystal structure of domains formed in bilayers that are composed of sphingomyelin (SM) and cholesterol (Chol), considered to be the main components of ordered lipid domains in cells. Structure measurements were derived by applying a new humidity control method, which we introduce here. The principle is to work close to the dew point of water and adjust the thickness of the condensed water layer on top of the sample through controlling the temperature differences between the sample and the humidified gas (Figure 1). The lipid bilayer sample is placed in a humidity chamber and covered with a thick layer of water. Evaporation is then initiated by increasing the temperature of the sample to 20 8C, thereby reducing the relative humidity (RH) above the bilayer to 42% (Figure 1). The RH is then increased gradually as the water layer thins, by cooling the sample to 6.7 8C. Under these conditions, the RH above the sample is 95.7 % and is thereafter kept rigorously constant. Bilayers are deposited by adapting Langmuir–Blodgett/Langmuir–Schaeffer techni[*] Prof. L. Leiserowitz Department of Materials and Interfaces Weizmann Institute of Science, 76100 Rehovot (Israel) Fax: (+ 972)89-344-138 E-mail: leslie.leiserowitz@weizmann.ac.il

Langmuir-Blodgett Films of a Functionalized Molecule with Cross-Sectional Mismatch Between Head and Tail

A functionalized surfactant has been investigated as floating monolayers by synchrotron x-ray diffraction and as bilayers transferred to solid supports by the Langmuir-Blodgett technique through atomic force microscopy. The transfer process is accompanied by an increase of the unit cell area (about 17 percent) and by an increase of the average domain diameter of nanometer-scale domains (about three times). The unit cell area of the floating monolayer corresponds to close packing of the head groups and a noncharacteristic packing of the tifted alkyl chains. The larger unit cell area of the bilayer film is consistent with a particular ordered packing of the alkyl chains, leaving free space for the head groups.