Hanna Wacklin

Composition and structure of mixed phospholipid supported bilayers formed by POPC and DPPC

In this paper we present a systematic study of the morphology and composition of supported lipid bilayers (SLBs) formed by vesicle fusion using a wide variety of surface sensitive techniques that give information about the lateral as well as vertical structure and bilayer fluidity. SLBs of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixtures at five different bulk vesicle compositions were formed in such a way that the phase separation boundaries were crossed. For all compositions studied, the SLBs were systematically enriched with POPC compared to the nominal vesicle composition. Nevertheless, gel-fluid domain coexistence was observed for SLB compositions in which phase separation was expected based on the bulk phase diagram. The probable causes for the compositional difference in the SLBs are discussed in terms of the phase behaviour of the mixture and its effect on the membrane formation process by vesicle fusion.

Formation of supported lipid bilayers by vesicle fusion: effect of deposition temperature.

We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.

Continuous Flow Atomic Force Microscopy Imaging Reveals Fluidity and Time-Dependent Interactions of Antimicrobial Dendrimer with Model Lipid Membranes

In this paper, an amphiphilic peptide dendrimer with potential applications against multi-resistant bacteria such as Staphylococcus aureus was synthesized and studied on model cell membranes. The combination of quartz crystal microbalance and atomic force microscopy imaging during continuous flow allowed for in situ monitoring of the very initial interaction processes and membrane transformations on longer time scales. We used three different membrane compositions of low and high melting temperature phospholipids to vary the membrane properties from a single fluid phase to a pure gel phase, while crossing the phase coexistence boundaries at room temperature. The interaction mechanism of the dendrimer was found to be time-dependent and to vary remarkably with the fluidity and coexistence of liquid-solid phases in the membrane. Spherical micelle-like dendrimer-lipid aggregates were formed in the fluid-phase bilayer and led to partial solubilization of the membrane, while in gel-phase membranes, the dendrimers caused areas of local depressions followed by redeposition of flexible lipid patches. Domain coexistence led to a sequence of events initiated by the formation of a ribbon-like network and followed by membrane solubilization via spherical aggregates from the edges of bilayer patches. Our results show that the dendrimer molecules were able to destroy the membrane integrity through different mechanisms depending on the lipid phase and morphology and shed light on their antimicrobial activity. These findings could have an impact on the efficacy of the dendrimers since lipid membranes in certain bacteria have transition temperatures very close to the host body temperature.

Fluid and highly curved model membranes on vertical nanowire arrays.

Sensing and manipulating living cells using vertical nanowire devices requires a complete understanding of cell behavior on these substrates. Changes in cell function and phenotype are often triggered by events taking place at the plasma membrane, the properties of which are influenced by local curvature. The nanowire topography can therefore be expected to greatly affect the cell membrane, emphasizing the importance of studying membranes on vertical nanowire arrays. Here, we used supported phospholipid bilayers as a model for biomembranes. We demonstrate the formation of fluid supported bilayers on vertical nanowire forests using self-assembly from vesicles in solution. The bilayers were found to follow the contours of the nanowires to form continuous and locally highly curved model membranes. Distinct from standard flat supported lipid bilayers, the high aspect ratio of the nanowires results in a large bilayer surface available for the immobilization and study of biomolecules. We used these bilayers to bind a membrane-anchored protein as well as tethered vesicles on the nanowire substrate. The nanowire-bilayer platform shown here can be expanded from fundamental studies of lipid membranes on controlled curvature substrates to the development of innovative membrane-based nanosensors.

Recent applications of synchrotron radiation and neutrons in the study of soft matter

ABSTRACT The broad range of applications of synchrotron and neutron scattering in the investigation of soft condensed matter is reviewed. Appropriate combinations of these techniques allow probing the structure and dynamics of these complex systems from sub-nm to micron size scales and picoseconds to seconds and longer time ranges. Applications include a myriad of systems such as polymers, colloids, surfactants, phospholipids, biological macromolecules and functional materials both in bulk and at interfaces. Most studies are performed in situ under the real thermodynamic state of the given system and large ensemble averaged information is readily obtained. The new generations of synchrotron and neutron sources open possibilities for investigating more complex soft matter systems in hitherto unexplored dynamical states.

Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation.

Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII-lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC-SM-Chol membranes in a manner identical to that of the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein-membrane interactions available from neutron reflection measurements.

Aligning Nanodiscs at the Air-Water Interface, a Neutron Reflectivity Study

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 Å with a surface coverage of 66 ± 4%. This layer is located about 15 Å below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 Å) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.

RNA and DNA association to zwitterionic and charged monolayers at the air-liquid interface.

The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA. In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology.

Molecular Dynamics Simulations and Neutron Reflectivity as an Effective Approach To Characterize Biological Membranes and Related Macromolecular Assemblies

In combination with other spectroscopy, microscopy, and scattering techniques, neutron reflectivity is a powerful tool to characterize biological systems. Specular reflection of neutrons provides structural information at the nanometer and subnanometer length scales, probing the composition and organization of layered materials. Currently, analysis of neutron reflectivity data involves several simplifying assumptions about the structure of the sample under study, affecting the extraction and interpretation of information from the experimental data. Computer simulations can be used as a source of structural and dynamic data with atomic resolution. We present a novel tool to compare the structural properties determined by neutron reflectivity experiments with those obtained from molecular simulations. This tool allows benchmarking the ability of molecular dynamics simulations to reproduce experimental data, but it also promotes unbiased interpretation of experimentally determined quantities. Two application examples are presented to illustrate the capabilities of the new tool. The first example is the generation of reflectivity profiles for a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer from molecular dynamics simulations using data from both atomistic and coarse-grained models, and comparison with experimentally measured data. The second example is the calculation of lipid volume changes with temperature and composition from all atoms simulations of single and mixed 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) bilayers.

Structure of DNA-Cationic Surfactant Complexes at Hydrophobically Modified and Hydrophilic Silica Surfaces as Revealed by Neutron Reflectometry

In this article, we discuss the structure and composition of mixed DNA-cationic surfactant adsorption layers on both hydrophobic and hydrophilic solid surfaces. We have focused on the effects of the bulk concentrations, the surfactant chain length, and the type of solid surface on the interfacial layer structure (the location, coverage, and conformation of the DNA and surfactant molecules). Neutron reflectometry is the technique of choice for revealing the surface layer structure by means of selective deuteration. We start by studying the interfacial complexation of DNA with dodecyltrimethylammonium bromide (DTAB) and hexadecyltrimethylammonium bromide (CTAB) on hydrophobic surfaces, where we show that DNA molecules are located on top of a self-assembled surfactant monolayer, with the thickness of the DNA layer and the surfactant-DNA ratio determined by the surface coverage of the underlying cationic layer. The surface coverages of surfactant and DNA are determined by the bulk concentration of the surfactant relative to its critical micelle concentration (cmc). The structure of the interfacial layer is not affected by the choice of cationic surfactant studied. However, to obtain similar interfacial structures, a higher concentration in relation to its cmc is required for the more soluble DTAB surfactant with a shorter alkyl chain than for CTAB. Our results suggest that the DNA molecules will spontaneously form a relatively dense, thin layer on top of a surfactant monolayer (hydrophobic surface) or a layer of admicelles (hydrophilic surface) as long as the surface concentration of surfactant is great enough to ensure a high interfacial charge density. These findings have implications for bioanalytical and nanotechnology applications, which require the deposition of DNA layers with well-controlled structure and composition.