Fusion Of Small Unilamellar Vesicles Research Articles

Membrane elasticity modulated by cholesterol in model of porcine eye lens-lipid membrane

Experimental evidence shows that the eye lens loses its elasticity dramatically with age. It has also been reported that the cholesterol (Chol) content in the eye lens fiber cell plasma membrane increases significantly with age. High Chol content leads to the formation of cholesterol bilayer domains (CBDs) in the lens membrane. The role of high Chol associated with lens elasticity is unclear. The purpose of this research is to investigate the membrane elasticity of the model of porcine lens-lipid (MPLL) membrane with increasing Chol content to elucidate the role of high Chol in lens membrane elasticity. In this study, we used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and area compressibility modulus (KA)) of the MPLL membrane with increasing Chol content where KA is the measure of membrane elasticity. We varied Chol concentration in Chol/MPLL membrane from 0 to ∼71 mol%. Supported Chol/MPLL membranes were prepared by fusion of small unilamellar vesicles (SUVs) on top of a flat mica surface. SUVs of the Chol/MPLL lipid mixture were prepared with the rapid solvent exchange method followed by probe-tip sonication. For the Chol/MPLL mixing ratio of 0, AFM image showed the formation of two distinct phases of the membrane, i.e., liquid-disordered phase (ld) and solid-ordered phase (so) membrane. However, with Chol/MPLL mixing ratio of 0.5 and above, only liquid-ordered phase (lo) membrane was formed. Also, two distinct breakthrough forces corresponding to ld and so were observed for Chol/MPLL mixing ratio of 0, whereas only one breakthrough force was observed for membranes with Chol/MPLL mixing ratio of 0.5 and above. No significant difference in the membrane surface roughness was measured with increasing Chol content for these membranes; however, breakthrough force and KA for lo membrane increased when Chol/MPLL mixing ratio was increased from 0.5 to 1. Interestingly above the Chol/MPLL mixing ratio of 1, both breakthrough force and KA decreased, indicating the formation of CBDs. Furthermore, these results showed that membrane elasticity increases at high Chol content, suggesting that high Chol content in lens membrane might be responsible for maintaining lens membrane elasticity.

Mixed Self-Assembled Monolayers with Terminal Deuterated Anchors: Characterization and Probing of Model Lipid Membrane Formation.

We describe herein a series of self-assembled monolayers (SAMs) on gold designed for adjustable tethering of model lipid membrane phases. The SAMs consist of deuterated aliphatic anchors, HS(CH2)15CONH(CH2CH2O)6CH2CONH-X, where X is either -(CD2)7CD3 or -(CD2)15CD3, dispersed in a stable matrix of protein-repellent molecules, HS(CH2)15CONHCH2CH2OH. The mixed SAMs with variable surface densities of the anchors are thoroughly characterized before and after adsorption of phospholipids by means of ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy (IRRAS). In all cases, the bottom portions of the mixed SAMs (i.e., the h-alkyl thiol segments of the molecules) are arranged in a highly ordered all-trans conformation stabilized by a network of lateral hydrogen bonds. The terminal portions of the anchors (the oligo(ethylene glycol) spacer and deuterated alkyl segments, respectively), however, possess less ordered conformations in the mixed composition regime. For the SAMs containing the longer anchors (-(CD2)15CD3), the contact angle and infrared data point toward partial phase segregation. These findings are in excellent agreement with molecular dynamics simulations by Schulze and Stein. Upon analysis in air, the IRRAS data also indicate strong interaction between the adsorbed phospholipid molecules and the d-alkyl tails of the mixed SAM constituents. In such assemblies are the alkyl tails of the phospholipids aligned perpendicularly with respect to the supporting substrate, regardless of the anchor length. We also probed the in situ formation of a tethered bilayer lipid membrane (tBLM) via fusion of small unilamellar vesicles (SUVs) on the characterized SAMs using a quartz crystal microbalance with dissipation monitoring. Our experiments show that SUVs fuse efficiently of the two mixed SAMs with and without a pre-adsorbed lipid layer. Owing to the defined molecular composition and phase behavior, our SAM platform is attractive for detailed studies of tBLM formation and cell mimetic applications.

SNARE-Mediated Transient Fusion of Liposomes to Supported Bilayers Probed by Two-Color pTIRFM

In membrane fusion opening of a fusion pore establishes the first connection between two compartments. During neurotransmitter or hormone release via exocytosis, fusion pores can transiently open and close repeatedly (flicker) before resealing (‘kiss-and-run’) or dilating irreversibly (full fusion). Pore dynamics regulate the amount and size of released cargo and determine the mode of vesicle recycling, yet what governs pore dynamics is poorly understood. We established an assay to monitor membrane fusion by polarized total internal reflection (pTIRF) microscopy with single molecule sensitivity and ∼15 ms time resolution in a biochemically well-defined system. In our assay, fusion of small unilamellar vesicles containing v-SNARE proteins (v-SUVs) fuse to a flat bilayer reconstituted with cognate t-SNAREs, supported on a soft polymer cushion (t-SBL). Exploiting the signal enhancement when a label transfers from the SUV to the SBL membrane in pTIRF, we could monitor the release kinetics of vesicular lipid labels into the SBL upon fusion. We found release times were much longer than would be expected for permanently open pores. Our sensitivity to single fluorescent lipids allowed us to calculate the lipid diffusivity and SUV size for each fusion event. This was used in a model we developed assuming the retardation was due to flickering pores, to estimate pore openness P0, the fraction of time a pore remains open during fusion. Increasing cholesterol levels increased P0. However, retardation of lipid release could also be due to steric hindrance of lipid diffusion through the walls of the fusion pore by SNARE proteins. To exclude this, we encapsulated soluble markers into the SUVs and simultaneously monitored lipid and contents release. Release of both lipid and soluble cargo started simultaneously. Interestingly, many pores resealed after partially releasing their contents.

PH Alters PEG-Mediated Fusion of Phosphatidylethanolamine-Containing Vesicles

Here, we examine the different mechanisms of poly(ethylene glycol)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPE)/sphingomyelin/cholesterol in a molar ratio of 35:30:15:20 at pH 7.4 versus pH 5. In doing so, we test the hypothesis that fusion of this lipid mixture should be influenced by differences in hydration of DOPE at these two pH values. An examination of the literature reveals that DOPE should be less hydrated at pH 5 (where influenza virus particles fuse with endosome membranes) than at pH 7.4 (where synaptic vesicles or HIV virus particles fuse with plasma membrane). Ensemble kinetic experiments revealed substantial differences in fusion of this plasma membrane mimetic system at these two pH values. The most dramatic difference was the observation of two intermediates at pH 5 but loss of one of these fusion intermediates at pH 7.4. Analysis of data collected at several temperatures also revealed that formation of the initial fusion intermediate (stalk) was favored at pH 7.4 due to increased activation entropy. Our observations support the hypothesis that the different negative intrinsic curvature of DOPE can account for different fusion paths and activation thermodynamics in steps of the fusion process at these two pH values. Finally, the effects of 2 mol % hexadecane on fusion at both pH values seemed to have similar origins for step 1 (promotion of acyl chain or hydrocarbon excursion into interbilayer space) and step 3 (reduction of interstice energy leading to expansion to a critical stalk radius). Different hexadecane effects on activation thermodynamics at these two pH values can also be related to altered DOPE hydration. The results support our kinetic model for fusion and offer insight into the critical role of phosphatidylethanolamine in fusion.

Wild-Type and Mutant Hemagglutinin Fusion Peptides Alter Bilayer Structure as Well as Kinetics and Activation Thermodynamics of Stalk and Pore Formation Differently: Mechanistic Implications

Viral fusion peptides are short N-terminal regions of type-1 viral fusion proteins that are critical for virus entry. Although the importance of viral fusion peptides in virus-cell membrane fusion is established, little is known about how they function. We report the effects of wild-type (WT) hemagglutinin (HA) fusion peptide and its G1S, G1V, and W14A mutants on the kinetics of poly(ethylene glycol)(PEG)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, and cholesterol (molar ratio of 35:30:15:20). Time courses of lipid mixing, content mixing, and content leakage were obtained using fluorescence assays at multiple temperatures and analyzed globally using either a two-step or three-step sequential ensemble model of the fusion process to obtain the rate constant and activation thermodynamics of each step. We also monitored the influence of peptides on bilayer interfacial order, acyl chain order, bilayer free volume, and water penetration. All these data were considered in terms of a recently published mechanistic model for the thermodynamic transition states for each step of the fusion process. We propose that WT peptide catalyzes Step 1 by occupying bilayer regions vacated by acyl chains that protrude into interbilayer space to form the Step 1 transition state. It also uniquely contributes a positive intrinsic curvature to hemi-fused leaflets to eliminate Step 2 and catalyzes Step 3 by destabilizing the highly stressed edges of the hemi-fused microstructures that dominate the ensemble of the intermediate state directly preceding fusion pore formation. Similar arguments explain the catalytic and inhibitory properties of the mutant peptides and support the hypothesis that the membrane-contacting fusion peptide of HA fusion protein is key to its catalytic activity.

Fabrication of GM3‐Enriched Sphingomyelin/Cholesterol Solid‐Supported Lipid Membranes on Au/SiO2 Plasmonic Substrates

The fabrication of solid-supported artificial lipid bilayers enriched with biological agents is an important step for acquiring more insights into their behavior from in vitro studies. In this work we demonstrate the formation of lipid artificial membranes enriched with ganglioside GM3, whose role in the cell proliferation and tumor angiogenesis is still an object of extensive investigation. The membranes are formed by fusion of small unilamellar vesicles (SUV) obtained from the sonication of multi-lamellar vesicles (MLV) formed from a hydrated mixture of GM3, brain sphingomyelin and cholesterol. The effective formation of SUV was confirmed by Dynamic light scattering (DLS) measurements. The recognition of the ganglioside in the biomimetic raft like biomembranes is accomplished via surface plasmon resonance (SPR) by exploiting the ganglioside affinity with wheat germ agglutinin (WGA). The affinity constant of the binding between the inglobated GM3 and WGA has been measured for three different GM3 molar concentrations. Beside the effective generation of GM3-enriched biomimetic membranes, our results indicate a decrease in the apparent WGA-GM3 dissociation constant at increasing GM3 concentrations up to 20 mol%, consistent with clustering effects of the ganglioside in the fabricated biomembranes.

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.

Tethered Bilayer Lipid Membranes on Mixed Self-Assembled Monolayers of a Novel Anchoring Thiol: Impact of the Anchoring Thiol Density on Bilayer Formation

Tethered bilayer lipid membranes (tBLMs) are designed on mixed self-assembled monolayers (SAMs) of a novel synthetic anchoring thiol, 2,3-di-o-palmitoylglycerol-1-tetraethylene glycol mercaptopropanoic acid ester (TEG-DP), and a new short dilution thiol molecule, tetraethylene glycol mercaptopropanoic acid ester (TEG). tBLM formation was accomplished by self-directed fusion of small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The influence of the dilution of the anchoring thiol molecule in the SAM on the vesicle fusion process and on the properties of the resulting tBLMs is studied. It is observed by quartz crystal microbalance that vesicle fusion is a one-step process for a pure TEG-DP SAM as well as for mixed SAMs containing a high concentration of the anchoring thiol. However, upon dilution of the anchoring thiol to moderate concentrations, this process is decelerated and possibly follows a pathway different from that observed on a pure TEG-DP SAM. Electrochemical impedance spectroscopy is used to qualitatively correlate the composition of the SAM to the electrical properties of the tBLM. In this paper we also delineate the necessity of a critical concentration of this anchoring TEG-DP thiol as a requisite for inducing the fusion of vesicles to form a tBLM.

Phospholipids as an alternative to direct covalent coupling: Surface functionalization of nanoporous alumina for protein recognition and purification

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75nm and depths of 3.5 or 10μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs’ lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin–DOPE containing POPC membrane, showing maximum protein coverage at 10mol% of biotin–DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14kDa) and ezrin (70kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS–PAGE.

A Fresh Look at Cochleate Cylinder by Freeze-Fracture Electron Microscopy: From Fusion Intermediate to Carrier of Antibiotics

Cochleate cylinders are tightly wrapped, micrometer-size tubes of concentric bilayers folded in a spiral configuration. They were discovered during Ca2+-induced fusion of small unilamellar vesicles made of phosphatidylserine [1]. Now they are used as platform technology for antigen delivery for vaccination and for oral drug delivery. Here we report the formation of cochleate cylinder by treating PS-free lipid films with cationic antimicrobial peptides without the requirement for divalent cations. The lipid films employed are mimicking the lipid pattern of cytoplasmic membranes of Gram negative bacteria. The antimicrobial peptides chosen contain the oligo-acyl-lysyl (OAK) sequence C12K-7α8. Due to their cationic nature they replace the divalent cations in the cochleate cylinder forming process. Furthermore, this new type of cochleate cylinder has been proven to encapsulate and transport traditional, essentially ineffective antibiotics such as erythromycin to resistant bacteria cells [2]. The double-function of OAK-based antimicrobial peptides in forming cochleate cylinder and promoting encapsulation and effective delivery of traditional antibiotics in/by these assemblies provides an interesting approach to overcome antibiotic resistance in bacteria.

Direct Simulation of Protein-Mediated Vesicle Fusion: Lung Surfactant Protein B

We simulated spontaneous fusion of small unilamellar vesicles mediated by lung surfactant protein B (SP-B) using the MARTINI force field. An SP-B monomer triggers fusion events by anchoring two vesicles and facilitating the formation of a lipid bridge between the proximal leaflets. Once a lipid bridge is formed, fusion proceeds via a previously described stalk – hemifusion diaphragm – pore-opening pathway. In the absence of protein, fusion of vesicles was not observed in either unbiased simulations or upon application of a restraining potential to maintain the vesicles in close proximity. The shape of SP-B appears to enable it to bind to two vesicles at once, forcing their proximity, and to facilitate the initial transfer of lipids to form a high-energy hemifusion intermediate. Our results may provide insight into more general mechanisms of protein-mediated membrane fusion, and a possible role of SP-B in the secretory pathway and transfer of lung surfactant to the gas exchange interface.

Fusogenic Activity of PLA2-IIA and SMase in PEG-Mediated Membrane Fusion

Phospholipase A2-IIA (PLA2-IIA) and sphingomyelinase (SMase) have been shown to induce fusion of membranes both in vivo and in vitro. The fusogenic potential of these two enzymes has been observed, for example, during neurotransmitter release. It is also believed that the products of hydrolysis of these two enzymes induce a reduction in the free energy cost of the fusion event. Assessment of the biophysical mechanisms by which these enzymes propel membrane fusion may provide relevant information for a variety of biological scenarios including neurotransmitter release. Poly(ethylene glycol)-(PEG-) mediated fusion of small unilamellar vesicles (SUVs) was studied in the presence of PLA2-IIA and SMase at varying temperatures between 17C and 40C. Lipid composition was dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), porcine brain sphingomyelin (SM), and cholesterol (CH) in a ratio of 35:30:15:20. Content mixing (CM), lipid mixing (LM) and leakage (L) time courses were obtained and globally fitted to a 3 or 4-state sequential vesicle fusion model introduced by Weinberg and Lentz (Weinberg and Lentz, Biophys. J., 2007, 92; 4012). Based on the global fits, rate constants for the transitions between states, and LM, CM and L occurrence probabilities are obtained. We observe that both LM and CM are enhanced by enzyme activity in SUVs. In addition, the exogenous addition of hydrolysis products also promotes LM and CM in the absence of the enzymes. A careful study of the temperature dependent plots is used to explore alterations in the energy barriers between intermediate states. These findings may shed light into the biological mechanisms wherein specific vesicle lipid structure and enzyme catalysis become interrelated to induce membrane fusion.

Lipid bilayers on topochemically structured planar colloidal crystals: a versatile platform for optical recording of membrane-mediated ion transport

Phospholipid membranes assembled on a planar colloidal crystal functionalized with a proton sensitive fluorescence probe offers a useful model system for a convenient, on-chip, parallel, and fluorescence based assay of membrane-mediated ion transport. The fusion of small unilamellar vesicles on surface derivatized hydrophilic/hydrophobic planar colloidal crystals results in a single lipid bilayer on the hydrophilic region and a monolayer on the hydrophobic region. We show that this composite structure exhibits a laterally continuous outer leaflet spanning the two mono- and bilayer morphologies, thereby providing an effective seal against ionic permeability. This is in contrast to the structure of lipids assembled on a patterned wettability planar coverslip where there a lipophobic “moat” formed at the junction between the mono- and the bilayer configurations prevents forming well-sealed membrane. By embedding a pH sensitive fluorescent probe, namely, fluorescein, within the colloidal crystal interstices, we find that the colloidal crystal supported membrane configuration provides a practical assay for optical characterization of membrane mediated ionic transport. We demonstrate the ability of our platform for the case of passive transport of protons across a fluid phospholipid bilayer. The membrane permeability coefficients derived, including those associated with the biphasic permeation mechanism, match well with those reported for vesicle based assays and confirm the quantitative optical transduction. We also illustrate the use of this experimental platform for parallel measurements such as may be useful for the characterization of stochastic transport and high throughput measurements.

Changes in molecular composition and packing during lipidmembrane reconstitution from phospholipid–surfactant micelles

Total internal reflection (TIR) Raman spectroscopy was used to investigate changes in the properties of phospholipid bilayers at the quartz–water interface during the process of membrane reconstitution from lipid–detergent micelles. The non-ionic detergent β-D-dodecyl maltoside (β-DDM) was used in 9 : 1 ratio to deuterated and non-deuterated dipalmitoylphosphatidyl choline (DPPC and d75-DPPC) to form detergent–lipid micelles. Exposure of the quartz surface to decreasing concentrations of such detergent–lipid solutions resulted in the formation of a d75-DPPC bilayer indistinguishable from that of a bilayer formed by fusion of small unilamellar vesicles of the pure lipid, indicating that the use of detergent micelles does not alter the packing or conformation of lipids, or leave a residue of surfactant in the bilayer. Polarised measurements of d75DPPC spectra in the C–H and C–D regions at each step of the process showed that the lipid surface density increases as the detergent is removed by rinsing and replaced by more lipid. The absence of shifts in the frequencies demonstrates that DPPC is in a gel-like conformation even at high relative surfactant fractions. The TIR geometry also allowed us to record high resolution spectra in the low frequency C–D bending region of d75-DPPC, which showed that the lipid molecules have an upright orientation with respect to the support surface.

Employing an Amphipathic Viral Peptide to Create a Lipid Bilayer on Au and TiO2

Supported lipid bilayers formed by the fusion of small unilamellar vesicles onto SiO2 or organic film-modified surfaces serve as model membranes in both scientific research and practical applications. Here, we describe the use of an amphipathic α-helical viral peptide derived from the hepatitis C virus NS5A protein (AH peptide) to destabilize the vesicles leading to lipid bilayer formation on gold and TiO2 solid substrates. Whereas previous researchers have been limited in their selection of surface materials for lipid biomembranes, the use of such peptides as destabilizing agents will allow the freedom to choose a broader variety of solid substrates to support planar bilayers. In particular, the favorable electrical properties of gold and the beneficial biocompatibility of TiO2 make these substrates attractive. The formation of model lipid bilayers supported on gold and TiO2 substrates can be utilized in many membrane-associated biological, physiological, or electrochemical applications with the advantag...

Molecular Dynamics Simulations of Lipid Vesicle Fusion in Atomic Detail

The fusion of a membrane-bounded vesicle with a target membrane is a key step in intracellular trafficking, exocytosis, and drug delivery. Molecular dynamics simulations have been used to study the fusion of small unilamellar vesicles composed of a dipalmitoyl-phosphatidylcholine (DPPC)/palmitic acid 1:2 mixture in atomic detail. The simulations were performed at 350–370 K and mimicked the temperature- and pH-induced fusion of DPPC/palmitic acid vesicles from experiments by others. To make the calculations computationally feasible, a vesicle simulated at periodic boundary conditions was fused with its periodic image. Starting from a preformed stalk between the outer leaflets of the vesicle and its periodic image, a hemifused state formed within 2 ns. In one out of six simulations, a transient pore formed close to the stalk, resulting in the mixing of DPPC lipids between the outer and the inner leaflet. The hemifused state was (meta)stable on a timescale of up to 11 ns. Forcing a single lipid into the interior of the hemifusion diaphragm induced the formation and expansion of a fusion pore on a nanosecond timescale. This work opens the perspective to study a wide variety of mesoscopic biological processes in atomic detail.

Carbon Nanotube Supported Single Phospholipid Bilayer

Single bilayer membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were formed on micron thin-films of hydrophilized carbon nanotubes (CNT) by fusion of small unilamellar vesicles. The structure of the membrane was investigated using neutron reflectivity (NR). The underlying thin film of CNT was formed by chemical vapor deposition (CVD) in the presence of Fe catalyst, followed by reaction with 5 M nitric acid to render the film hydrophilic. We demonstrate that this platform lends support to homogeneous and continuous bilayer membranes that have promising applications in the fields of biomaterials, biosensors, and biophysics.

Diffusion measurements of water, ubiquinone and lipid bilayer inside a cylindrical nanoporous support: A stimulated echo pulsed-field gradient MAS-NMR investigation

Stimulated echo pulsed-field gradient 1H magic angle spinning NMR has been used to investigate the mobility of water, ubiquinone and tethered phospholipids, components of a biomimetic model membrane. The diffusion constant of water corresponds to an isotropic motion in a cylinder. When the lipid bilayer is obtained after the fusion of small unilamellar vesicles, the extracted value of lipid diffusion indicates unrestricted motion. The cylindrical arrangement of the lipids permits a simplification of data analysis since the normal bilayer is perpendicular to the gradient axis. This feature leads to a linear relation between the logarithm of the attenuation of the signal intensity and a factor depending on the gradient strength, for lipids covering the inner wall of aluminium oxide nanopores as well as for lipids adsorbed on a polymer sheet rolled into a cylinder. The effect of the bilayer formation on water diffusion has also been observed. The lateral diffusion coefficient of ubiquinone is in the same order of magnitude as the lipid lateral diffusion coefficient, in agreement with its localization within the bilayer.

Tethered or Adsorbed Supported Lipid Bilayers in Nanotubes Characterized by Deuterium Magic Angle Spinning NMR Spectroscopy

2H solid-state NMR experiments were performed under magic angle spinning on lipid bilayers oriented into nanotubes arrays, as a new method to assess the geometrical arrangement of the lipids. Orientational information is obtained from the intensities of the spinning sidebands. The lipid bilayers are formed by fusion of small unilamellar vesicles of DMPC-d54 inside a nanoporous anodic aluminum oxide, either by direct adsorption on the support or by tethering through a streptavidin/biotin linker. The results support that the quality of the lipid bilayers alignment is clearly in favor of the tethering rather than an adsorbed strategy.

Neutron Reflectivity Study of Lipid Membranes Assembled on Ordered Nanocomposite and Nanoporous Silica Thin Films

Single bilayer membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were formed on ordered nanocomposite and nanoporous silica thin films by fusion of small unilamellar vesicles. The structure of these membranes was investigated using neutron reflectivity. The underlying thin films were formed by evaporation induced self-assembly to obtain periodic arrangements of silica and surfactant molecules in the nanocomposite thin films, followed by photocalcination to oxidatively remove the organics and render the films nanoporous. We show that this platform affords homogeneous and continuous bilayer membranes that have promising applications as model membranes and sensors.