Supported lipid bilayers at skeletonized surfaces for the study of transmembrane proteins

Skeletonized zirconium phosphonate surfaces are used to support planar lipid bilayers and are shown to be viable substrates for studying transmembrane proteins. The skeletonized surfaces provide space between the bilayer and the solid support to enable protein insertion and avoid denaturation. The skeletonized zirconium octadecylphosphonate surfaces were prepared using Langmuir-Blodgett techniques by mixing octadecanol with octadecylphosphonic acid. After zirconation of the transferred monolayer, rinsing the coating with organic solvent removes the octadecanol, leaving holes in the film ranging from ～50 to ～500 nm in diameter, depending on the octadecanol content. Upon subsequent deposition of a lipid bilayer, either by vesicle fusion or by Langmuir-Blodgett/Langmuir-Schaefer techniques, the lipid assemblies span the holes providing reservoirs beneath the bilayer. The viability of the supported bilayers as model membranes for transmembrane proteins was demonstrated by examining two approaches for incorporating the proteins. The BK channel protein inserts directly into a preformed bilayer on the skeletonized surface, in contrast to a bilayer on a nonskeletonized film, for which the protein associates only weakly. As a second approach, the integrin α(5)β(1) was reconstituted in lipid vesicles, and its inclusion in supported bilayers on the skeletonized surface was achieved by vesicle fusion. The integrin retains its ability to recognize the extracellular matrix protein fibronectin when supported on the skeletonized film, again in contrast to the response if the bilayer is supported on a nonskeletonized film.     

Effect of freeze-thawing on lipid bilayer-protected gold nanoparticles

In this study, varieties of lipid bilayer-protected gold nanoparticles (AuNPs) were synthesized through a simple wet chemical method, and then the effect of freeze-thawing on the as-prepared AuNPs was investigated. The freeze-thawing process induced fusion or fission of lipid bilayers tethered on the AuNPs. The UV-vis spectra and transmission electron microscopy experiments revealed that the disruption of lipid bilayer structures on the nanoparticles led to the fusion or aggregation of AuNPs. The role of freeze-thawing in the evolution of lipid bilayer-protected AuNPs was studied. The addition of adequate sucrose, a well-known cryoprotectant, effectively prevented the fusion or aggregation of lipid bilayer-protected AuNPs undergoing the freeze-thawing process. The possible mechanism of sucrose preserving the integrity of the lipid bilayer-protected AuNPs was also discussed.     

Gold Nanoparticle Assemblies on Surfaces: Reactivity Tuning through Capping‐Layer and Cross‐Linker Design

The immobilization of metal nanoparticles (NPs) with molecular control over their organization is challenging. Herein, we report the formation of molecularly cross‐linked AuNP assemblies using a layer‐by‐layer approach. We observed four types of assemblies: 1) small aggregates of individual AuNPs, 2) large aggregates of individual AuNPs, 3) networks of fused AuNPs, and 4) gold islands. Interestingly, these assemblies with the different cross‐linkers and capping layers represent different stages in the complete fusion of AuNPs to afford islands of continuous gold. We demonstrate that the stability toward fusion of the nanoparticles of the on‐surface structures can be controlled by the reactivity of the cross‐linkers and the hydrophilicity/hydrophobicity of the nanoparticles.     

Didodecyldimethylammonium bromide lipid bilayer-protected gold nanoparticles: synthesis, characterization, and self-assembly

Didodecyldimethylammonium bromide (DDAB) lipid bilayer-protected gold nanoparticles (AuNPs), which were stable and hydrophilic, were synthesized by in situ reduction of HAuCl(4) with NaBH(4) in an aqueous medium in the presence of DDAB. As-prepared nanoparticles were characterized by UV-vis spectra, transmission electron microscopy, dynamic light scattering analysis, and X-ray photoelectron spectroscopy. All these data supported the formation of AuNPs. Fourier transform infrared spectroscopy (FTIR) and differential thermal analysis/thermogravimetric analysis data revealed that DDAB existed in a bilayer structure formed on the particle surface, resulting in a positively charged particle surface. The FTIR spectra also indicated that the DDAB bilayer coated on the surface of AuNPs was probably in the ordered gel phase with some end-gauche defects. On the basis of electrostatic interactions between such AuNPs and anionic polyelectrolyte poly(sodium 4-styrenesulfonate) (PSS), we successfully fabricated (PSS/AuNP)(n)() multilayers on a cationic polyelectrolyte poly(ethylenimine) coated indium tin oxide substrate via the layer-by-layer self-assembly technique and characterized as-formed multilayers with UV-vis spectra and atomic force microscopy.     

Free Energy of Bare and Capped Gold Nanoparticles Permeating through a Lipid Bilayer

Herein, we study the permeation free energy of bare and octane‐thiol‐capped gold nanoparticles (AuNPs) translocating through a lipid membrane. To investigate this, we have pulled the bare and capped AuNPs from bulk water to the membrane interior and estimated the free energy cost. The adsorption of the bare AuNP on the bilayer surface is energetically favorable but further loading inside it requires energy. However, the estimated free‐energy barrier for loading the capped AuNP into the lipid membrane is much higher compared to bare AuNP. We also demonstrate the details of the permeation process of bare and capped AuNPs. Bare AuNP induces the curvature in the lipid membrane whereas capped AuNP creates an opening in the interacting monolayer and get inserted into the membrane. The insertion of capped AuNP induces a partial unzipping of the lipid bilayer, which results in the ordering of the local lipids interacting with the nanoparticle. However, bare AuNP disrupts the lipid membrane by pushing the lipid molecules inside the membrane. We also analyze pore formation due to the insertion of capped AuNP into the membrane, which results in water molecules penetrating the hydrophobic region.     

Laminar order within Langmuir-Blodgett multilayers from phospholipid and myelin basic protein: a neutron reflectivity study

Multilayers consisting of negatively charged phospholipid DMPA and myelin basic protein (MBP) were assembled by Langmuir-Blodgett deposition of floating Langmuir monolayers from the air/water interface to solid substrates. Protein/lipid samples were obtained by binding MBP from the aqueous subphase to the phospholipid monolayers before deposition. The vertical organization of these model membranes (i.e., with organization perpendicular to the substrate surface) was investigated in detail by neutron reflectivity measurements, and the internal distribution of water molecules was determined from the change of contrast after in-situ H2O/D2O exchange. The multilayers were well ordered, with repeating lipid bilayers as fundamental structural unit. MBP was inserted in between adjacent lipid headgroups, such as in the natural myelin membrane. Water molecules in the multilayers were present mainly in the lipid headgroup and protein slab. On exposition of the pure lipid multilayers to a dry atmosphere, a reduction of the bilayer spacing was determined, whereas the global lamellar order was not affected. In contrast, drying of the protein/lipid multilayers induced degradation of the laminar order. The data demonstrate that ordered Langmuir-Blodgett multilayers are versatile model systems for studying how competing interactions between lipid, protein, water, and ions affect the global organization of such multilamellar lipid/protein assemblies. Here, the water molecules were found to be a necessary mediator to maintain the laminar order in a multilayer from DMPA and myelin basic protein.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Ion current calculations based on three dimensional Poisson-Nernst-Planck theory for a cyclic peptide nanotube

Ion current calculations based on Poisson-Nernst-Planck (PNP) theory are performed for a synthetic cyclic peptide nanotube that consists of eight or ten cyclo[(-L-Trp-D-Leu-)4] embedded in a lipid bilayer membrane to investigate the ion transport properties of the nanotube. To explore systems with arbitrary geometries, three-dimensional PNP theory is implemented using a finite difference method. The influence of dipolar lipid molecules on the ion currents is also examined by turning on or off the charges of the lipid dipoles in dipalmitoylphosphatidylcholine (DPPC). Comparisons between the calculated and experimentally measured ion currents show that the PNP approach agrees well with the measurements at low ion concentrations but overestimates the currents at higher concentrations. Concentration profiles reveal the selectivity of the peptide nanotube to cations, which is attributed to the negatively charged carbonyl oxygens inside the nanotube. The dominant cation and the minimum anion concentrations inside the cyclic peptide nanotube suggest that these cyclic peptide nanotubes can be employed as ion sensors. In the case of the polar DPPC bilayer, smaller currents are obtained in the calculation. The variation of current with polarity of the lipids implies that both polar and nonpolar lipid bilayer membranes can be utilized to regulate ion currents in the peptide nanotube and other ion channels. Strengths and limitations of the PNP theory are also discussed.     

Potential of mean force and pKa profile calculation for a lipid membrane-exposed arginine side chain

The issue of ionizable protein side chains interacting with lipid membranes has been the focus of much attention since the proposal of the paddle model of voltage-gated ion channels, which suggested multiple arginine (Arg) side chains may move through the hydrocarbon core of a lipid membrane. Recent cell biology experiments have also been interpreted to suggest that these side chains would face only small free energy penalties to cross membranes, challenging a long-standing view in membrane biophysics. Here, we employ side chain analog and transmembrane helix models to determine the free energy of an Arg side chain, as a function of protonation state, across a membrane. We observe high free energy barriers for both the charged and neutral states that would prohibit lipid-exposed movement. The mechanisms for charged and neutral Arg transport are, however, very different, with the neutral state experiencing simple dehydration, whereas the charged state experiences a complex mechanism involving connections to the bilayer interfaces that deform the local membrane structure. We employ special methods to ensure sampling of these interfacial connections and decompose the free energy to shed light on the mechanisms. These deformations are found to preferentially stabilize the protonated form, such that the Arg side chain remains almost exclusively charged inside the membrane, with a pKa shift of 相似文献   

Role of lipid charge in organization of water/lipid bilayer interface: insights via computer simulations

Anionic unsaturated lipid bilayers represent suitable model systems that mimic real cell membranes: they are fluid and possess a negative surface charge. Understanding of detailed molecular organization of water-lipid interfaces in such systems may provide an important insight into the mechanisms of proteins' binding to membranes. Molecular dynamics (MD) of full-atom hydrated lipid bilayers is one of the most powerful tools to address this problem in silico. Unfortunately, wide application of computational methods for such systems is limited by serious technical problems. They are mainly related to correct treatment of long-range electrostatic effects. In this study a physically reliable model of an anionic unsaturated bilayer of 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) was elaborated and subjected to long-term MD simulations. Electrostatic interactions were treated with two different algorithms: spherical cutoff function and particle-mesh Ewald summation (PME). To understand the role of lipid charge in the system behavior, similar calculations were also carried out for zwitterionic bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). It was shown that, for the charged DOPS bilayer, the PME protocol performs much better than the cutoff scheme. In the last case a number of artifacts in the structural organization of the bilayer were observed. All of them were attributed to inadequate treatment of electrostatic interactions of lipid headgroups with counterions. Electrostatic properties, along with structural and dynamic parameters, of both lipid bilayers were investigated. Comparative analysis of the MD data reveals that the water-lipid interface of the DOPC bilayer is looser than that for DOPS. This makes possible deeper penetration of water molecules inside the zwitterionic (DOPC) bilayer, where they strongly interact with carbonyls of lipids. This can lead to thickening of the membrane interface in zwitterionic as compared to negatively charged bilayers.     

Amphiphilic Nanoparticles Control the Growth and Stability of Lipid Bilayers with Open Edges

Molecular amphiphiles self‐assemble in polar media to form ordered structures such as micelles and vesicles essential to a broad range of industrial and biological processes. Some of these architectures such as bilayer sheets, helical ribbons, and hollow tubules are potentially useful but inherently unstable owing to the presence of open edges that expose the hydrophobic bilayer core. Here, we describe a strategy to stabilize open bilayer structures using amphiphilic nanoparticle surfactants that present mixtures of hydrophilic and hydrophobic ligands on their surface. We observe that these particles bind selectively to the open edge of bilayer membranes to stabilize otherwise transient amphiphile assemblies. We show how such particles can precisely control the size of lipid tubules, how they can inhibit the formation of undesirable assemblies such as gallstone precursors, and how they can stabilize free‐floating lipid microdiscs.     

Local anesthetics facilitate ion transport across lipid planar bilayer membranes under an electric field: dependence on type of lipid bilayer

In order to elucidate the role of structural change of lipid membrane bilayer in the mode of action of local anesthetic, we studied the effects of local anesthetics, charged tetracaine and uncharged benzocaine, on ion permeability across various lipid planar bilayers (PC, mixed PC/PS (4/1, mol/mol); mixed PC/PE (1/1, mol/mol); mixed PC/SM (4/1, mol/mol)) under a constant applied voltage. The membrane conductances increased in the order of PC  PC/PS ≤ PC/SM  PC/PE. When the constant voltage of −100 or −70 mV was applied through the lipid bilayer membranes in the presence of positively charged tetracaine, the fluctuating current pulses with the large amplitude generated, but not appeared in the absence of tetracaine. The addition of uncharged benzocaine generated the fluctuating currents with the small amplitude. Both charged tetracaine and uncharged benzocaine facilitated electrophoretically the transport of small ions such as KCl in the buffer solution through the fluctuating pores in the lipid bilayer membranes formed by interaction with the local anesthetic under the negative applied membrane potential. The current pulses also contained actual transport of charged tetracaine together with the transport of the small ions. The amplitude and the duration time of the electrical current generated by adding the local anesthetics were dependent on the type of the lipid, the applied voltage and its voltage polarity.     

Probing organization and communication at layered interfaces

We have investigated the local organization intrinsic to a variety of interfacial structures, by both electrochemical and spectroscopic means. Our focus has been on the design and construction of biomimetic interfaces, where a lipid bilayer or a hybrid bilayer membrane can be bound to an interface. The goal of this work is ultimately to create an interface on a transducer surface that can support an enzyme in its active form. To this point, we have examined the extent of organization that is achievable in monolayers that will be used to bind bilayer structures to a transducer surface. Our electrochemical data point to the important role of the substrate surface in determining adlayer organization. We have also investigated the fluidity and structural heterogeneity of lipid bilayers using time-resolved and steady state fluorescence spectroscopy. Our data point to the highly interactive nature of lipid bilayer constituents, where perturbations introduced to one region have significant consequences on other regions of the bilayer. Such information is directly relevant to the existence and properties of lipid raft structures in both model and biological bilayers.     

A Halogen-Bond-Driven Artificial Chloride-Selective Channel Constructed from 5-Iodoisophthalamide-based Molecules

Despite considerable emphasis on advancing artificial ion channels, progress is constrained by the limited availability of small molecules with the necessary attributes of self-assembly and ion selectivity. In this study, a library of small molecules based on 5-haloisophthalamide and a non-halogenated isophthalamide were examined for their ion transport properties across the lipid bilayer membranes, and the finding demonstrates that the di-hexyl-substituted 5-iodoisophthalamide derivative exhibits the highest level of activity. Furthermore, it was established that the highest active compound facilitates the selective chloride transport that occurs via an antiport-mediated mechanism. The crystal structure of the compound unveils a distinctive self-assembly of molecules, forming a zig-zag channel pore that is well-suited for the permeation of anions. Planar bilayer conductance measurements proved the formation of chloride selective channels. A molecular dynamics simulation study, relying on the self-assembled component derived from the crystal structure, affirmed the paramount significance of intermolecular hydrogen bonding in the formation of supramolecular barrel-rosette structures that span the bilayer. Furthermore, it was demonstrated that the transport of chloride across the lipid bilayer membrane is facilitated by the synergistic effects of halogen bonding and hydrogen bonding within the channel.     

Structure and phase transformations of DPPC lipid bilayers in the presence of nanoparticles: insights from coarse-grained molecular dynamics simulations

In this article, we investigate fluid-gel transformations of a DPPC lipid bilayer in the presence of nanoparticles, using coarse-grained molecular dynamics. Two types of nanoparticles are considered, specifically a 3 nm hydrophobic nanoparticle located in the core of the bilayer and a 6 nm charged nanoparticle located at the interface between the bilayer and water phase. Both negatively and positively charged nanoparticles at the bilayer interface are investigated. We demonstrate that the presence of all types of nanoparticles induces disorder effects in the structure of the lipid bilayer. These effects are characterized using computer visualization of the gel phase in the presence of nanoparticles, radial distribution functions, and order parameters. The 3 nm hydrophobic nanoparticle immersed in the bilayer core and the positively charged nanoparticle at the bilayer surface have no effect on the temperature of the fluid-gel transformation, compared to the bulk case. Interestingly, a negatively charged hydrophobic nanoparticle located at the surface of the bilayer causes slight shift of the fluid-gel transformation to a lower temperature, compared to the bulk bilayer case.     

Permanent gold nanoparticle coatings on polyelectrolyte multilayer modified capillaries for open-tubular capillary electrochromatography

This paper reports on a new strategy to coat fused silica capillaries through ionic adsorption of gold nanoparticles (AuNPs) on a polyelectrolyte multilayer (PEM) modified capillary wall. The coating was constructed in situ by alternating rinses with positively charged poly(diallydimethylammonium chloride), negatively charged poly(sodium-4-styrenesulfonate), and positively charged AuNPs. After self-assembly of n-octadecanethiol onto the surface of AuNPs, the modified capillary was investigated as a new medium for the separation of neutral analytes and proteins in open-tubular capillary electrochromatography (OT-CEC). The surface coverage of the capillary wall was increased using the high density of AuNPs which were dynamically capped with 4-dimethylaminopyridine (DMAP). The chromatographic performance of the column coated with positively charged AuNPs was remarkably improved compared with a column modified with negatively charged AuNPs. The coating was robust over more than 810 runs in this study and also showed high stability against 0.01 M NaOH, 0.01 M HCl, and electrolyte concentrations up to 70 mM. The run-to-run, day-to-day, and capillary-to-capillary reproducibilities of electroosmotic flow were satisfying with relative standard deviation values of less than 1% in all cases. The AuNP-coated PEM modified capillary column not only showed good performance for neutral analytes but also was suitable for the analysis of both basic and acidic proteins.     

Colloidal titration of aqueous zirconium solutions with poly(vinyl sulfate) by potentiometric endpoint detection using a toluidine blue selective electrode.

Zirconium oxy-salts were hydrolyzed to form positively charged polymer or cluster species in acidic solutions. The zirconium hydrolyzed polymer was found to react with a negatively charged polyelectrolyte, such as poly(vinyl sulfate), and to form a stoichiometric polyion complex. Thus, colloidal titration with poly(vinyl sulfate) was applied to measure the zirconium concentration in an acidic solution by using a Toluidine Blue selective plasticized poly(vinyl chloride) membrane electrode as a potentiometric end-point detecting device. The determination could be performed with 1% of the relative standard deviation. The colloidal titration stoichiometry at pH < or = 2 was one mol of zirconium per equivalent mol of poly(vinyl sulfate).     

On the nature of antimicrobial activity: a model for protegrin-1 pores

We conducted over 150 ns of simulation of a protegrin-1 octamer pore in a lipid bilayer composed of palmitoyloleoyl-phosphatidylethanolamine (POPE) and palmitoyloleoyl-phosphatidylglycerol (POPG) lipids mimicking the inner membrane of a bacterial cell. The simulations improve on a model of a pore proposed from recent NMR experiments and provide a coherent understanding of the molecular mechanism of antimicrobial activity. Although lipids tilt somewhat toward the peptides, the simulated protegrin-1 pore more closely follows the barrel-stave model than the toroidal-pore model. The movement of ions is investigated through the pore. The pore selectively allows negatively charged chloride ions to pass through at an average rate of one ion every two nanoseconds. Only two events are observed of sodium ions crossing through the pore. The potential of mean force is calculated for the water and both ion types. It is determined that the chloride ions move through the pore with ease, similarly to the water molecules with the exception of a zone of restricted movement midway through the pore. In bacteria, ions moving through the pore will compromise the integrity of the transmembrane potential. Without the transmembrane potential as a countermeasure, water will readily flow inside the higher osmolality cytoplasm. We determine that the diffusivity of water through a single PG-1 pore is sufficient to cause fast cell death by osmotic lysis.     

Mechanism of unassisted ion transport across membrane bilayers

To establish how charged species move from water to the nonpolar membrane interior and to determine the energetic and structural effects accompanying this process, we performed molecular dynamics simulations of the transport of Na+ and Cl- across a lipid bilayer located between two water lamellae. The total length of molecular dynamics trajectories generated for each ion was 10 ns. Our simulations demonstrate that permeation of ions into the membrane is accompanied by the formation of deep, asymmetric thinning defects in the bilayer, whereby polar lipid head groups and water penetrate the nonpolar membrane interior. Once the ion crosses the midplane of the bilayer the deformation "switches sides"; the initial defect slowly relaxes, and a defect forms in the outgoing side of the bilayer. As a result, the ion remains well solvated during the process; the total number of oxygen atoms from water and lipid head groups in the first solvation shell remains constant. A similar membrane deformation is formed when the ion is instantaneously inserted into the interior of the bilayer. The formation of defects considerably lowers the free energy barrier to transfer of the ion across the bilayer and, consequently, increases the permeabilities of the membrane to ions, compared to the rigid, planar structure, by approximately 14 orders of magnitude. Our results have implications for drug delivery using liposomes and peptide insertion into membranes.     

Formation of polyelectrolyte multilayer architectures with embedded DMPC studied in situ by neutron reflectometry

The coupling of lipid molecules to polymer components in a planar biomimetic model membrane made of a lipid bilayer (dimyristoylphosphatidylcholine) supported by polyelectrolyte multilayers is studied. The polyelectrolyte support was prepared by layer-by-layer deposition of positively charged poly(allylamine hydrochloride) (PAH) and negatively charged poly(sodium 4-styrenesulfonate) (PSS). Two polymer sample terminations were considered: positively charged (PAH-terminated) and negatively charged (PSS-terminated). Neutron reflectometry studies showed that, whereas positively charged samples did not favor the deposition of lipid, negatively charged samples allowed the deposition of a lipid bilayer with a thickness of approximately 5 nm. In the latter case, formation of polyelectrolyte layers after the deposition of the lipid layer was also possible.