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Sample records for adjacent lipid bilayers

  1. An engineered dimeric protein pore that spans adjacent lipid bilayers

    PubMed Central

    Mantri, Shiksha; Sapra, K. Tanuj; Cheley, Stephen; Sharp, Thomas H.; Bayley, Hagan

    2013-01-01

    The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin (αHL) pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7)2, was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. We show that one of two β barrels of (α7)2 can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. PMID:23591892

  2. Immobilized lipid-bilayer materials

    DOEpatents

    Sasaki, Darryl Y.; Loy, Douglas A.; Yamanaka, Stacey A.

    2000-01-01

    A method for preparing encapsulated lipid-bilayer materials in a silica matrix comprising preparing a silica sol, mixing a lipid-bilayer material in the silica sol and allowing the mixture to gel to form the encapsulated lipid-bilayer material. The mild processing conditions allow quantitative entrapment of pre-formed lipid-bilayer materials without modification to the material's spectral characteristics. The method allows for the immobilization of lipid membranes to surfaces. The encapsulated lipid-bilayer materials perform as sensitive optical sensors for the detection of analytes such as heavy metal ions and can be used as drug delivery systems and as separation devices.

  3. Nanoparticle-lipid bilayer interactions studied with lipid bilayer arrays.

    PubMed

    Lu, Bin; Smith, Tyler; Schmidt, Jacob J

    2015-05-01

    The widespread environmental presence and commercial use of nanoparticles have raised significant health concerns as a result of many in vitro and in vivo assays indicating toxicity of a wide range of nanoparticle species. Many of these assays have identified the ability of nanoparticles to damage cell membranes. These interactions can be studied in detail using artificial lipid bilayers, which can provide insight into the nature of the particle-membrane interaction through variation of membrane and solution properties not possible with cell-based assays. However, the scope of these studies can be limited because of the low throughput characteristic of lipid bilayer platforms. We have recently described an easy to use, parallel lipid bilayer platform which we have used to electrically investigate the activity of 60 nm diameter amine and carboxyl modified polystyrene nanoparticles (NH2-NP and COOH-NP) with over 1000 lipid bilayers while varying lipid composition, bilayer charge, ionic strength, pH, voltage, serum, particle concentration, and particle charge. Our results confirm recent studies finding activity of NH2-NP but not COOH-NP. Detailed analysis shows that NH2-NP formed pores 0.3-2.3 nm in radius, dependent on bilayer and solution composition. These interactions appear to be electrostatic, as they are regulated by NH2-NP surface charge, solution ionic strength, and bilayer charge. The ability to rapidly measure a large number of nanoparticle and membrane parameters indicates strong potential of this bilayer array platform for additional nanoparticle bilayer studies.

  4. Nanoparticle-lipid bilayer interactions studied with lipid bilayer arrays

    NASA Astrophysics Data System (ADS)

    Lu, Bin; Smith, Tyler; Schmidt, Jacob J.

    2015-04-01

    The widespread environmental presence and commercial use of nanoparticles have raised significant health concerns as a result of many in vitro and in vivo assays indicating toxicity of a wide range of nanoparticle species. Many of these assays have identified the ability of nanoparticles to damage cell membranes. These interactions can be studied in detail using artificial lipid bilayers, which can provide insight into the nature of the particle-membrane interaction through variation of membrane and solution properties not possible with cell-based assays. However, the scope of these studies can be limited because of the low throughput characteristic of lipid bilayer platforms. We have recently described an easy to use, parallel lipid bilayer platform which we have used to electrically investigate the activity of 60 nm diameter amine and carboxyl modified polystyrene nanoparticles (NH2-NP and COOH-NP) with over 1000 lipid bilayers while varying lipid composition, bilayer charge, ionic strength, pH, voltage, serum, particle concentration, and particle charge. Our results confirm recent studies finding activity of NH2-NP but not COOH-NP. Detailed analysis shows that NH2-NP formed pores 0.3-2.3 nm in radius, dependent on bilayer and solution composition. These interactions appear to be electrostatic, as they are regulated by NH2-NP surface charge, solution ionic strength, and bilayer charge. The ability to rapidly measure a large number of nanoparticle and membrane parameters indicates strong potential of this bilayer array platform for additional nanoparticle bilayer studies.The widespread environmental presence and commercial use of nanoparticles have raised significant health concerns as a result of many in vitro and in vivo assays indicating toxicity of a wide range of nanoparticle species. Many of these assays have identified the ability of nanoparticles to damage cell membranes. These interactions can be studied in detail using artificial lipid bilayers, which

  5. Cholesterol's location in lipid bilayers

    DOE PAGES

    Marquardt, Drew; Kučerka, Norbert; Wassall, Stephen R.; Harroun, Thad A.; Katsaras, John

    2016-04-04

    It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group is in themore » vicinity of the lipid–water interface. In this study we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies.« less

  6. Lipid bilayers on nano-templates

    DOEpatents

    Noy, Aleksandr; Artyukhin, Alexander B.; Bakajin, Olgica; Stoeve, Pieter

    2009-08-04

    A lipid bilayer on a nano-template comprising a nanotube or nanowire and a lipid bilayer around the nanotube or nanowire. One embodiment provides a method of fabricating a lipid bilayer on a nano-template comprising the steps of providing a nanotube or nanowire and forming a lipid bilayer around the polymer cushion. One embodiment provides a protein pore in the lipid bilayer. In one embodiment the protein pore is sensitive to specific agents

  7. Cholera toxin interactions with lipid bilayers.

    PubMed

    Tosteson, M T; Tosteson, D C; Rubnitz, J

    1980-01-01

    The purpose of the experiments described in this paper was to assess the binding of cholera toxin to bilayers containing its receptor, the monosialoganglioside, GMl. The assay was based on the fact that GMl confers on the bilayer a negative surface charge. The magnitude of this surface charge was estimated by measuring the electrical conductance (G) of the bilayers exposed to nonactin-K+ under conditions where G is directly proportional to the potassium concentration in the aqueous solutions immediatey adjacent to the membrane surface. When bilayers were formed from mixtures of GMl and glycerolmonooleate (GMO), it was found that the molar ratio of the lipids in the bilayer was the same as that in the membrane forming solution. It was further found that cholera toxin or the binding subunit of the toxin (choleragenoid) bind to GMO bilayers containing GMl (but not to GMO bilayers containing phosphatidyl serine or disialoganglioside GDla). The value of the apparent dissociation constant for the binding of choleragen to its receptor was found to be 10(-11) M, comparable to values found in intact cells.

  8. Cholesterol Perturbs Lipid Bilayers Nonuniversally

    SciTech Connect

    Pan Jianjun; Mills, Thalia T.; Tristram-Nagle, Stephanie; Nagle, John F.

    2008-05-16

    Cholesterol is well known to modulate the physical properties of biomembranes. Using modern x-ray scattering methods, we have studied the effects of cholesterol on the bending modulus K{sub C}, the thickness D{sub HH}, and the orientational order parameter S{sub xray} of lipid bilayers. We find that the effects are different for at least three classes of phospholipids characterized by different numbers of saturated hydrocarbon chains. Most strikingly, cholesterol strongly increases K{sub C} when both chains of the phospholipid are fully saturated but not at all when there are two monounsaturated chains.

  9. Lipid mobility in supported lipid bilayers by single molecule tracking

    NASA Astrophysics Data System (ADS)

    Kohram, Maryam; Shi, Xiaojun; Smith, Adam

    2015-03-01

    Phospholipid bilayers are the main component of cell membranes and their interaction with biomolecules in their immediate environment is critical for cellular functions. These interactions include the binding of polycationic polymers to lipid bilayers which affects many cell membrane events. As an alternative method of studying live cell membranes, we assemble a supported lipid bilayer and investigate its binding with polycationic polymers in vitro by fluorescently labeling the molecules of the supported lipid bilayer and tracking their mobility. In this work, we use single molecule tracking total internal reflection fluorescence microscopy (TIRF) to study phosphatidylinositol phosphate (PIP) lipids with and without an adsorbed polycationic polymer, quaternized polyvinylpyridine (QPVP). Individual molecular trajectories are obtained from the experiment, and a Brownian diffusion model is used to determine diffusion coefficients through mean square displacements. Our results indicate a smaller diffusion coefficient for the supported lipid bilayers in the presence of QPVP in comparison to its absence, revealing that their binding causes a decrease in lateral mobility.

  10. Droplet Microfluidics for Artificial Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Punnamaraju, Srikoundinya; Steckl, Andrew

    2012-02-01

    Droplet interface bilayer is a versatile approach that allows formation of artificial lipid bilayer membrane at the interface of two lipid monolayer coated aqueous droplets in a lipid filled oil medium. Versatility exists in the form of voltage control of DIB area, ability of forming networks of DIBs, volume control of droplets and lipid-oil, and ease of reformation. Significant effect of voltage on the area and capacitance of DIB as well as DIB networks are characterized using simultaneous optical and electrical recordings. Mechanisms behind voltage-induced effects on DIBs are investigated. Photo induced effect on the DIB membrane porosity is obtained by incorporating UVC-sensitive photo-polymerizable lipids in DIB. Photo-induced effects can be extended for in-vitro studies of triggered release of encapsulated contents across membranes. A droplet based low voltage digital microfluidic platform is developed to automate DIB formation, which could potentially be used for forming arrays of lipid bilayer membranes.

  11. Masking apertures enabling automation and solution exchange in sessile droplet lipid bilayers.

    PubMed

    Portonovo, Shiva A; Schmidt, Jacob

    2012-02-01

    Reconstitution of ion channels and transmembrane proteins in planar lipid bilayer membranes allow for their scientific study in highly controlled environments. Recent work with lipid bilayers formed from mechanically joined monolayers has shown their potential for wider technological application, including automation and parallelization. However, bilayer areas are highly sensitive to variations in mechanical position and the bilayers themselves cannot withstand significant perfusion of adjacent solutions. Toward this end, here we describe use of an aperture that masks the monolayer contact area, enabling formation of highly consistent bilayer areas and significantly reducing their variation with changes in relative position of the monolayers. Further, use of the aperture enables flow of solution adjacent to the bilayer without rupture or significant change in bilayer area. The device design is scalable and compatible with SBS standard instrumentation and automation technology, potentially enabling its use for rapid, parallel automated measurements of ion channels for large scale scientific studies and pharmaceutical screening.

  12. Lipid bilayers covalently anchored to carbon nanotubes.

    PubMed

    Dayani, Yasaman; Malmstadt, Noah

    2012-05-29

    The unique physical and electrical properties of carbon nanotubes make them an exciting material for applications in various fields such as bioelectronics and biosensing. Due to the poor water solubility of carbon nanotubes, functionalization for such applications has been a challenge. Of particular need are functionalization methods for integrating carbon nanotubes with biomolecules and constructing novel hybrid nanostructures for bionanoelectronic applications. We present a novel method for the fabrication of dispersible, biocompatible carbon nanotube-based materials. Multiwalled carbon nanotubes (MWCNTs) are covalently modified with primary amine-bearing phospholipids in a carbodiimide-activated reaction. These modified carbon nanotubes have good dispersibility in nonpolar solvents. Fourier transform infrared (FTIR) spectroscopy shows peaks attributable to the formation of amide bonds between lipids and the nanotube surface. Simple sonication of lipid-modified nanotubes with other lipid molecules leads to the formation of a uniform lipid bilayer coating the nanotubes. These bilayer-coated nanotubes are highly dispersible and stable in aqueous solution. Confocal fluorescence microscopy shows labeled lipids on the surface of bilayer-modified nanotubes. Transmission electron microscopy (TEM) shows the morphology of dispersed bilayer-coated MWCNTs. Fluorescence quenching of lipid-coated MWCNTs confirms the bilayer configuration of the lipids on the nanotube surface, and fluorescence anisotropy measurements show that the bilayer is fluid above the gel-to-liquid transition temperature. The membrane protein α-hemolysin spontaneously inserts into the MWCNT-supported bilayer, confirming the biomimetic membrane structure. These biomimetic nanostructures are a promising platform for the integration of carbon nanotube-based materials with biomolecules.

  13. Molecular dynamics simulations of unsaturated lipid bilayers

    NASA Astrophysics Data System (ADS)

    Rabinovich, Alexander L.; Balabaev, Nikolay K.

    2001-02-01

    Molecular dynamics simulations were carried out for bilayers of lipid molecules having stearic acid (C18:0) chain in position '3-D' (using the nomenclature of M. Sundaralingam, 1972) and fatty acid chain C18:0, C18:1(omega 9), C18:2(omega 6), C18:3(omega 3), C20:4(omega 6) or C22:6(omega 3) in position '2-D'. To investigate the properties of the bilayers two models were considered. In the first model, the simulation cells of the bilayers consisted of 96 phosphatidylcholine (PC) molecules and 2304 water molecules: 48 lipid molecules per layer and 24 H2O molecules per lipid. The water was modeled by explicit TIP3P water molecules. In the second model, the head group of the lipid molecules was treated as an effective sphere -- diacylglycerolipids (DGs) were considered, the interface of each monolayer was modeled by a flat surface; no water molecules were present explicitly. The bilayers consisted of 48 X 2 equals 96 glycerolipids arranged in a rectangular simulation cell. Various properties of the bilayers -- the C-H bond order parameter -SCH profiles of the hydrocarbon tails, the root-mean-square values of the positional fluctuations of the lipid chain carbons, mass density distributions of lipid molecules and water along the normals were investigated.

  14. Molecular dynamics simulations of unsaturated lipid bilayers

    NASA Astrophysics Data System (ADS)

    Rabinovich, Alexander L.; Balabaev, Nikolay K.

    2000-02-01

    Molecular dynamics simulations were carried out for bilayers of lipid molecules having stearic acid (C18:0) chain in position '3-D' (using the nomenclature of M. Sundaralingam, 1972) and fatty acid chain C18:0, C18:1(omega 9), C18:2(omega 6), C18:3(omega 3), C20:4(omega 6) or C22:6(omega 3) in position '2-D'. To investigate the properties of the bilayers two models were considered. In the first model, the simulation cells of the bilayers consisted of 96 phosphatidylcholine (PC) molecules and 2304 water molecules: 48 lipid molecules per layer and 24 H2O molecules per lipid. The water was modeled by explicit TIP3P water molecules. In the second model, the head group of the lipid molecules was treated as an effective sphere -- diacylglycerolipids (DGs) were considered, the interface of each monolayer was modeled by a flat surface; no water molecules were present explicitly. The bilayers consisted of 48 X 2 equals 96 glycerolipids arranged in a rectangular simulation cell. Various properties of the bilayers -- the C-H bond order parameter -SCH profiles of the hydrocarbon tails, the root-mean-square values of the positional fluctuations of the lipid chain carbons, mass density distributions of lipid molecules and water along the normals were investigated.

  15. Supported lipid bilayer/carbon nanotube hybrids

    NASA Astrophysics Data System (ADS)

    Zhou, Xinjian; Moran-Mirabal, Jose M.; Craighead, Harold G.; McEuen, Paul L.

    2007-03-01

    Carbon nanotube transistors combine molecular-scale dimensions with excellent electronic properties, offering unique opportunities for chemical and biological sensing. Here, we form supported lipid bilayers over single-walled carbon nanotube transistors. We first study the physical properties of the nanotube/supported lipid bilayer structure using fluorescence techniques. Whereas lipid molecules can diffuse freely across the nanotube, a membrane-bound protein (tetanus toxin) sees the nanotube as a barrier. Moreover, the size of the barrier depends on the diameter of the nanotube-with larger nanotubes presenting bigger obstacles to diffusion. We then demonstrate detection of protein binding (streptavidin) to the supported lipid bilayer using the nanotube transistor as a charge sensor. This system can be used as a platform to examine the interactions of single molecules with carbon nanotubes and has many potential applications for the study of molecular recognition and other biological processes occurring at cell membranes.

  16. Method of fabricating lipid bilayer membranes on solid supports

    NASA Technical Reports Server (NTRS)

    Cho, Nam-Joon (Inventor); Frank, Curtis W. (Inventor); Glenn, Jeffrey S. (Inventor); Cheong, Kwang Ho (Inventor)

    2012-01-01

    The present invention provides a method of producing a planar lipid bilayer on a solid support. With this method, a solution of lipid vesicles is first deposited on the solid support. Next, the lipid vesicles are destabilized by adding an amphipathic peptide solution to the lipid vesicle solution. This destabilization leads to production of a planar lipid bilayer on the solid support. The present invention also provides a supported planar lipid bilayer, where the planar lipid bilayer is made of naturally occurring lipids and the solid support is made of unmodified gold or titanium oxide. Preferably, the supported planar lipid bilayer is continuous. The planar lipid bilayer may be made of any naturally occurring lipid or mixture of lipids, including, but not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinsitol, cardiolipin, cholesterol, and sphingomyelin.

  17. Viscoelastic deformation of lipid bilayer vesicles.

    PubMed

    Wu, Shao-Hua; Sankhagowit, Shalene; Biswas, Roshni; Wu, Shuyang; Povinelli, Michelle L; Malmstadt, Noah

    2015-10-01

    Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic.

  18. Supported Lipid Bilayer/Carbon Nanotube Hybrids

    NASA Astrophysics Data System (ADS)

    Zhou, Xinjian; Moran-Mirabal, Jose; Craighead, Harold; McEuen, Paul

    2007-03-01

    We form supported lipid bilayers on single-walled carbon nanotubes and use this hybrid structure to probe the properties of lipid membranes and their functional constituents. We first demonstrate membrane continuity and lipid diffusion over the nanotube. A membrane-bound tetanus toxin protein, on the other hand, sees the nanotube as a diffusion barrier whose strength depends on the diameter of the nanotube. Finally, we present results on the electrical detection of specific binding of streptavidin to biotinylated lipids with nanotube field effect transistors. Possible techniques to extract dynamic information about the protein binding events will also be discussed.

  19. Multi-Stacked Supported Lipid Bilayer Micropatterning through Polymer Stencil Lift-Off.

    PubMed

    Zhu, Yujie; Negmi, Ahmed; Moran-Mirabal, Jose

    2015-01-01

    Complex multi-lamellar structures play a critical role in biological systems, where they are present as lamellar bodies, and as part of biological assemblies that control energy transduction processes. Multi-lamellar lipid layers not only provide interesting systems for fundamental research on membrane structure and bilayer-associated polypeptides, but can also serve as components in bioinspired materials or devices. Although the ability to pattern stacked lipid bilayers at the micron scale is of importance for these purposes, limited work has been done in developing such patterning techniques. Here, we present a simple and direct approach to pattern stacked supported lipid bilayers (SLBs) using polymer stencil lift-off and the electrostatic interactions between cationic and anionic lipids. Both homogeneous and phase-segregated stacked SLB patterns were produced, demonstrating that the stacked lipid bilayers retain lateral diffusivity. We demonstrate patterned SLB stacks of up to four bilayers, where fluorescence resonance energy transfer (FRET) and quenching was used to probe the interactions between lipid bilayers. Furthermore, the study of lipid phase behaviour showed that gel phase domains align between adjacent layers. The proposed stacked SLB pattern platform provides a robust model for studying lipid behaviour with a controlled number of bilayers, and an attractive means towards building functional bioinspired materials or devices. PMID:26343733

  20. Multi-Stacked Supported Lipid Bilayer Micropatterning through Polymer Stencil Lift-Off

    PubMed Central

    Zhu, Yujie; Negmi, Ahmed; Moran-Mirabal, Jose

    2015-01-01

    Complex multi-lamellar structures play a critical role in biological systems, where they are present as lamellar bodies, and as part of biological assemblies that control energy transduction processes. Multi-lamellar lipid layers not only provide interesting systems for fundamental research on membrane structure and bilayer-associated polypeptides, but can also serve as components in bioinspired materials or devices. Although the ability to pattern stacked lipid bilayers at the micron scale is of importance for these purposes, limited work has been done in developing such patterning techniques. Here, we present a simple and direct approach to pattern stacked supported lipid bilayers (SLBs) using polymer stencil lift-off and the electrostatic interactions between cationic and anionic lipids. Both homogeneous and phase-segregated stacked SLB patterns were produced, demonstrating that the stacked lipid bilayers retain lateral diffusivity. We demonstrate patterned SLB stacks of up to four bilayers, where fluorescence resonance energy transfer (FRET) and quenching was used to probe the interactions between lipid bilayers. Furthermore, the study of lipid phase behaviour showed that gel phase domains align between adjacent layers. The proposed stacked SLB pattern platform provides a robust model for studying lipid behaviour with a controlled number of bilayers, and an attractive means towards building functional bioinspired materials or devices. PMID:26343733

  1. Hydrodynamic trapping of molecules in lipid bilayers

    PubMed Central

    Jönsson, Peter; McColl, James; Clarke, Richard W.; Ostanin, Victor P.; Jönsson, Bengt; Klenerman, David

    2012-01-01

    In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the hydrodynamic drag forces arising from a flow through a conical pipette with a tip radius of 1–1.5 μm, placed approximately 1 μm above the investigated SLB. This results in a localized forcefield that acts on molecules protruding from the SLB, yielding a hydrodynamic trap with a size approximately given by the size of the pipette tip. We demonstrate this concept by trapping the protein streptavidin, bound to biotin receptors in the SLB. It is also shown how static and kinetic information about the intermolecular interactions in the lipid bilayer can be obtained by relating how the magnitude of the hydrodynamic forces affects the accumulation of protein molecules in the trap. PMID:22699491

  2. Graphene-Templated Supported Lipid Bilayer Nanochannels.

    PubMed

    Li, Wan; Chung, Jean K; Lee, Young Kwang; Groves, Jay T

    2016-08-10

    The use of patterned substrates to impose geometrical restriction on the lateral mobility of molecules in supported lipid membranes has found widespread utility in studies of cell membranes. Here, we template-pattern supported lipid membranes with nanopatterned graphene. We utilize focused ion beam milling to pattern graphene on its growth substrate, then transfer the patterned graphene to fresh glass substrates for subsequent supported membrane formation. We observe that graphene functions as an excellent lateral diffusion barrier for supported lipid bilayers. Additionally, the observed diffusion dynamics of lipids in nanoscale graphene channels reveal extremely low boundary effects, a common problem with other materials. We suggest this is attributable to the ultimate thinness of graphene. PMID:27362914

  3. Engineering Lipid Bilayer Membranes for Protein Studies

    PubMed Central

    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

    2013-01-01

    Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

  4. Electrophoretic Measurements of Lipid Charges in Supported Bilayers

    PubMed Central

    Poyton, Matthew F.; Cremer, Paul S.

    2013-01-01

    While electrophoresis in lipid bilayers has been performed since the 1970’s, the technique has until now been unable to accurately measure the charge on lipids and proteins within the membrane based on drift velocity measurements. Part of the problem is caused by the use of the Einstein-Smoluchowski equation to estimate the electrophoretic mobility of such species. The source of the error arises from the fact that a lipid head group is typically smaller than the Debye length of the adjacent aqueous solution in most electrophoresis experiments. Instead, the Henry equation can more accurately predict the electrophoretic mobility at sufficient ionic strength. This was done for three dye-labeled lipids with different sized head groups and a charge on each lipid of −1. Also, the charge was measured as a function of pH for two titratable lipids that were fluorescently labeled. Finally, it was shown that the Henry equation also has difficulties measuring the correct lipid charge at salt concentrations below 5 mM, where electroosmotic forces are more significant. PMID:24191728

  5. Functional One-Dimensional Lipid Bilayers on Carbon Nanotube Templates

    SciTech Connect

    Artyukhin, A; Shestakov, A; Harper, J; Bakajin, O; Stroeve, P; Noy, A

    2004-07-23

    We present one-dimensional (1-D) lipid bilayer structures that integrate carbon nanotubes with a key biological environment-phospholipid membrane. Our structures consist of lipid bilayers wrapped around carbon nanotubes modified with a hydrophilic polymer cushion layer. Despite high bilayer curvature, the lipid membrane maintains its fluidity and can sustain repeated damage-recovery cycles. We also present the first evidence of spontaneous insertion of pore-forming proteins into 1-D lipid bilayers. These structures could lead to the development of new classes of biosensors and bioelectronic devices.

  6. Protein-lipid interactions in bilayer membranes: a lattice model.

    PubMed

    Pink, D A; Chapman, D

    1979-04-01

    A lattice model has been developed to study the effects of intrinsic membrane proteins upon the thermodynamic properties of a lipid bilayer membrane. We assume that only nearest-neighbor van der Waals and steric interactions are important and that the polar group interactions can be represented by effective pressure-area terms. Phase diagrams, the temperature T(0), which locates the gel-fluid melting, the transition enthalpy, and correlations were calculated by mean field and cluster approximations. Average lipid chain areas and chain areas when the lipid is in a given protein environment were obtained. Proteins that have a "smooth" homogeneous surface ("cholesterol-like") and those that have inhomogeneous surfaces or that bind lipids specifically were considered. We find that T(0) can vary depending upon the interactions and that another peak can appear upon the shoulder of the main peak which reflects the melting of a eutectic mixture. The transition enthalpy decreases generally, as was found before, but when a second peak appears departures from this behavior reflect aspects of the eutectic mixture. We find that proteins have significant nonzero probabilities for being adjacent to one another so that no unbroken "annulus" of lipid necessarily exists around a protein. If T(0) does not increase much, or decreases, with increasing c, then lipids adjacent to a protein cannot all be all-trans on the time scale (10(-7) sec) of our system. Around a protein the lipid correlation depth is about one lipid layer, and this increases with c. Possible consequences of ignoring changes in polar group interactions due to clustering of proteins are discussed.

  7. Repetitive formation of optically-observable planar lipid bilayers by rotating chambers on a microaperture.

    PubMed

    Tomoike, Fumiaki; Tonooka, Taishi; Osaki, Toshihisa; Takeuchi, Shoji

    2016-07-01

    Optical observation of a planar lipid bilayer is an effective method of lipid bilayer characterization. However, previous methods for optically observable lipid bilayer formation are unsuitable for repetitive formation of lipid bilayers. In this paper, we propose a system that facilitates repetitive formation of horizontal lipid bilayers via mechanical rotation of the rotating part. We show that multiple bilayers can be observed within a short period, and that the electrical and optical characteristics of a bilayer can be analyzed simultaneously. PMID:27256329

  8. Self-assembled lipid bilayer materials

    DOEpatents

    Sasaki, Darryl Y.; Waggoner, Tina A.; Last, Julie A.

    2005-11-08

    The present invention is a self-assembling material comprised of stacks of lipid bilayers formed in a columnar structure, where the assembly process is mediated and regulated by chemical recognition events. The material, through the chemical recognition interactions, has a self-regulating system that corrects the radial size of the assembly creating a uniform diameter throughout most of the structure. The materials form and are stable in aqueous solution. These materials are useful as structural elements for the architecture of materials and components in nanotechnology, efficient light harvesting systems for optical sensing, chemical processing centers, and drug delivery vehicles.

  9. Investigating the Structure of Multicomponent Gel-Phase Lipid Bilayers.

    PubMed

    Hartkamp, Remco; Moore, Timothy C; Iacovella, Christopher R; Thompson, Michael A; Bulsara, Pallav A; Moore, David J; McCabe, Clare

    2016-08-23

    Single- and multicomponent lipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), isostearyl isostearate, and heptadecanoyl heptadecanoate in the gel phase are studied via molecular dynamics simulations. It is shown that the structural properties of multicomponent bilayers can deviate strongly from the structures of their single-component counterparts. Specifically, the lipid mixtures are shown to adopt a compact packing by offsetting the positioning depths at which different lipid species are located in the bilayer. This packing mechanism affects the area per lipid, the bilayer height, and the chain tilt angles and has important consequences for other bilayer properties, such as interfacial hydrogen bonding and bilayer permeability. In particular, the simulations suggest that bilayers containing isostearyl isostearate or heptadecanoyl heptadecanoate are less permeable than pure 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine or DSPC bilayers. Furthermore, hydrogen-bond analysis shows that the residence times of lipid-water hydrogen bonds depend strongly on the bilayer composition, with longer residence times for bilayers that have a higher DSPC content. The findings illustrate and explain the fundamental differences between the properties of single- and multicomponent bilayers. PMID:27558724

  10. Lipid bilayer vesicle generation using microfluidic jetting.

    PubMed

    Coyne, Christopher W; Patel, Karan; Heureaux, Johanna; Stachowiak, Jeanne; Fletcher, Daniel A; Liu, Allen P

    2014-01-01

    Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies(1,2). Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation(3). The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting. PMID:24637415

  11. Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

    PubMed Central

    Coyne, Christopher W.; Patel, Karan; Heureaux, Johanna; Stachowiak, Jeanne; Fletcher, Daniel A.; Liu, Allen P.

    2014-01-01

    Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting. PMID:24637415

  12. Lipid bilayer vesicle generation using microfluidic jetting.

    PubMed

    Coyne, Christopher W; Patel, Karan; Heureaux, Johanna; Stachowiak, Jeanne; Fletcher, Daniel A; Liu, Allen P

    2014-02-21

    Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies(1,2). Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation(3). The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

  13. Structure and Orientational Texture of Self-Organizing Lipid Bilayers

    SciTech Connect

    Watkins, E. B.; Miller, C. E.; Majewski, J.; Mulder, D. J.; Kuhl, T. L.

    2009-06-12

    The structure of single supported dipalmitoyl-phosphatidylcholine bilayers prepared by vesicle fusion or Langmuir-Blodgett-Schaeffer (LBS) deposition techniques was characterized by x-ray reflectivity and grazing incidence diffraction in bulk water. LBS bilayers display symmetric leaflets similar to monolayer structures, while vesicle fusion yields more inhomogeneous bilayers. Diffraction establishes that lipids are always coupled across the bilayer even when leaflets are deposited independently and suggests the existence of orientational texture.

  14. Automatable lipid bilayer formation for ion channel studies

    NASA Astrophysics Data System (ADS)

    Poulos, Jason L.; Bang, Hyunwoo; Jeon, Tae-Joon; Schmidt, Jacob J.

    2008-08-01

    Transmembrane proteins and ion channels are important drug targets and have been explored as single molecule sensors. For these proteins to function normally they must be integrated within lipid bilayers; however, the labor and skill required to create artificial lipid bilayers have the limited the possible applications utilizing these proteins. In order to reduce the complexity and cost of lipid bilayer formation and measurement, we have modified a previously published lipid bilayer formation technique using mechanically contacted monolayers so that the process is automatable, requiring minimal operator input. Measurement electronics are integrated with the fluid handling system, greatly reducing the time and operator feedback characteristically required of traditional bilayer experiments. To demonstrate the biological functionality of the resultant bilayers and the system's capabilities as a membrane platform, the ion channel gramicidin A was incorporated and measured with this system.

  15. Asymmetric heat transfer from nanoparticles in lipid bilayers

    NASA Astrophysics Data System (ADS)

    Potdar, Dipti; Sammalkorpi, Maria

    2015-12-01

    Here, we use molecular dynamics simulations to characterize the heat transfer properties of lipid bilayer - gold nanoparticle systems in which the nanoparticle acts as a heat source. The focus is on dipalmitoylphosphatidylcholine (DPPC) lipid bilayers and thiolated alcohol and alkyl functionalized nanoparticles as prototype hydrophilic and hydrophobic nanoparticles. We find hydrophilic nanoparticles which are partly in contact with the surrounding water environment are more efficient in transferring heat to the system than hydrophobic ones which reside surrounded by the membrane. This is because of the hydrogen bonding capability of the hydroxy pentanethiol and the more efficient heat conductivity through water than the lipid bilayer. Additionally, we find the heat conductance is strongly asymmetric and has a discontinuity between the bilayer leaflets. In total, the findings provide understanding on heat transport from localized heat sources in lipid bilayers and could bear significance, e.g., in engineering and controlling photoactivated triggering of liposomal systems.

  16. Detection of bilayer lipid with graphene nanoribbon

    NASA Astrophysics Data System (ADS)

    Akbari, Elnaz; Buntat, Zolkafle; Afroozeh, Abdolkarim; Zeinalinezhad, Alireza; Nilashi, Mehrbakhsh

    2015-09-01

    Single-layer graphene consists of sp 2-bonded carbon atoms arranged in a two-dimensional (2D) hexagonal lattice comprising a thin layer of single carbon atoms. Owing to its special characteristics including electrical, physical, and optical properties, graphene is considered more suitable for sensor applications than other materials. Moreover, it is possible to produce biosensors using electrolyte-gated field-effect transistors based on graphene (GFETs) to identify the alterations in charged lipid membrane properties. This paper illustrates how membrane thickness and electrical charge can result in a monolayer GFET, with emphasis on conductance variation. It is proposed that the thickness and electrical charge of the lipid bilayer are functions of carrier density, and equations relating these suitable control parameters were derived. Adaptive neuro fuzzy inference system (ANFIS) has been incorporated to obtain other model for conductance characteristic. The comparison between the analytical models and ANFIS with the experimental data extracted from previous work show an acceptable agreement. [Figure not available: see fulltext.

  17. Polyunsaturated Fatty Acids in Lipid Bilayers and Tubules

    NASA Astrophysics Data System (ADS)

    Hirst, Linda S.; Yuan, Jing; Pramudya, Yohannes; Nguyen, Lam T.

    2007-03-01

    Omega-3 polyunsaturated fatty acids (PUFAs) are found in a variety of biological membranes and have been implicated with lipid raft formation and possible function, typical molecules include DHA (Docosahexanoic Acid) and AA (Alphalinoleic Acid) which have been the focus of considerable attention in recent years. We are interested in the phase behavior of these molecules in the lipid bilayer. The addition of lipid molecules with polyunsaturated chains has a clear effect on the fluidity and curvature of the membrane and we investigate the effects the addition of polyunsaturated lipids on bilayer structure and tubule formation. Self-assembled cylindrical lipid tubules have attracted considerable attention because of their interesting structures and potential technological applications. Using x-ray diffraction techniques, Atomic Force Microscopy and confocal fluorescence imaging, both symmetric and mixed chain lipids were incorporated into model membranes and the effects on bilayer structure and tubule formation investigated.

  18. Effect of benzyl alcohol on lipid bilayers. A comparisons of bilayer systems.

    PubMed Central

    Ebihara, L; Hall, J E; MacDonald, R C; McIntosh, T J; Simon, S A

    1979-01-01

    The effect of the small anesthetic molecule, benzyl alcohol, on the structure of various bilayer system has been studied by optical, electrical, and x-ray diffraction techniques. We find that the modifications in bilayer thickness caused by benzyl alcohol differ dramatically for planar (or black lipid) bilayers containing solvent, planar bilayers containing little or no solvent, and vesicular bilayers. Benzyl alcohol increases the thickness of planar bilayers containing n-alkane solvents, yet decreases the thickness of "solvent-free" planar bilayers. The effect of benzyl alcohol on vesicular bilayers below the phase transition temperature also depends on whether solvent is present in the bilayers. Without solvent, gel-state bilayers are reduced in thickness by benzyl alcohol, whereas in the presence of solvent, the thickness is unchanged. Above the phase transition temperature, benzyl alcohol has no measurable effect on vesicular bilayer thickness, whether solvent is present or not. These results indicate that different model membrane systems respond quite differently to a particular anesthetic. PMID:263698

  19. Intercalation of small hydrophobic molecules in lipid bilayers containing cholesterol

    SciTech Connect

    Worcester, D.L.; Hamacher, K.; Kaiser, H.; Kulasekere, R.; Torbet, J.

    1994-12-31

    Partitioning of small hydrophobic molecules into lipid bilayers containing cholesterol has been studied using the 2XC diffractometer at the University of Missouri Research Reactor. Locations of the compounds were determined by Fourier difference methods with data from both deuterated and undeuterated compounds introduced into the bilayers from the vapor phase. Data fitting procedures were developed for determining how well the compounds were localized. The compounds were found to be localized in a narrow region at the center of the hydrophobic layer, between the two halves of the bilayer. The structures are therefore intercalated structures with the long axis of the molecules in the plane of the bilayer.

  20. Microporous device for local electric recordings on model lipid bilayers

    NASA Astrophysics Data System (ADS)

    Kaufeld, Theresa; Steinem, Claudia; Schmidt, Christoph F.

    2015-01-01

    A powerful approach for characterizing lipid membranes and embedded proteins is the reconstitution of model lipid bilayers. The extreme fragility of 5 nm thick bilayers is a challenge for device design and requires a trade off of stability against accessibility. We here present a microporous lab-on-chip device that allows us to form stable, solvent-free lipid bilayers from giant unilamellar vesicles (GUVs) in a geometry that provides a unique set of access possibilities. The device is constructed around a micro-fabricated silicon chip with clusters of 1 µm-diameter pores and provides optical access to the lipid bilayers for high-NA epifluorescence imaging. At the same time, solvent exchange is possible on both sides of the lipid bilayer. Complete coverage can be achieved with GUVs, so that voltages can be applied across the lipid bilayer and single-channel currents can be measured using external or integrated silver/silver chloride electrodes. We describe the micro-fabrication by standard cleanroom techniques and the characterization of the device by atomic force microscopy, scanning electron microscopy and impedance spectroscopy. In proof-of-concept experiments we demonstrate that the device is capable of low-noise, single-ion-channel recordings. Electronic Supplementary Information (ESI) available: See DOI: 10.1039/b000000x/

  1. Fabrication of a planar zwitterionic lipid bilayer on titanium oxide.

    PubMed

    Cho, Nam-Joon; Frank, Curtis W

    2010-10-19

    There is great demand to fabricate planar phospholipid bilayers on biocompatible materials. The preferred method of forming bilayers on these substrates is the spontaneous adsorption and rupture of phospholipid vesicles. However, in the case of titanium oxide, model vesicles composed solely of zwitterionic phospholipids do not follow this self-assembly pathway under physiological conditions, prompting the use of complex bilayer materials and less-facile methods. Herein, we report a novel pH-based strategy for fabricating zwitterionic bilayers on titanium oxide in a simple and robust manner. Depending on the pH conditions under which lipid vesicles adsorb onto titanium oxide, quartz crystal microbalance-dissipation (QCM-D) monitoring demonstrated that the self-assembly pathway can in fact result in planar bilayer formation. The pH of the solution could then be adjusted to physiological levels with no effect on the mass and viscoelastic properties of the bilayer. Moreover, fluorescence recovery after photobleaching (FRAP) measurements indicated a high degree of lateral lipid diffusivity within the bilayer at physiological pH, commensurate with its role as a cell membrane mimic. Compared to existing protocols, this strategy permits the fabrication of a more diverse array of planar bilayers on titanium oxide by tuning the self-assembly pathway of lipid vesicle adsorption onto solid substrates.

  2. Effect of insulin on lipid bilayer viscoelasticity.

    PubMed

    Hianik, T; Zórad, S; Kavecanský, J; Macho, L

    1987-04-01

    Changes in the Young elasticity modulus in perpendicular direction to the membrane surface E perpendicular, in the coefficient of dynamic viscosity eta, in the electric capacitance C, in the surface charge U1, in the conductivity g and in the coefficient of non-linearity beta of current-voltage characteristic caused by insulin were studied in bilayer lipid membranes (BLM) prepared from a mixture of egg lecithin and cholesterol (4:1, w/w) in n-heptane. Even relatively small concentrations of insulin in electrolyte (ci approximately 4.8 x 10(-11) mol/l) caused a diminution in parameters E perpendicular and eta. Negative surface charge emerged on the membrane due to the insulin absorption, and U1 gradually increased depending on the concentration of the hormone in the electrolyte. Addition of insulin was also followed by an increase in membrane conductivity and affected the value of the coefficient of non-linearity beta of current-voltage characteristic. The effect of insulin on the BLM structure was discussed on the basis of the results obtained.

  3. Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

    PubMed Central

    Needham, D; Nunn, R S

    1990-01-01

    Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the

  4. Intermonolayer Friction and Surface Shear Viscosity of Lipid Bilayer Membranes

    PubMed Central

    den Otter, W. K.; Shkulipa, S. A.

    2007-01-01

    The flow behavior of lipid bilayer membranes is characterized by a surface viscosity for in-plane shear deformations, and an intermonolayer friction coefficient for slip between the two leaflets of the bilayer. Both properties have been studied for a variety of coarse-grained double-tailed model lipids, using equilibrium and nonequilibrium molecular dynamics simulations. For lipids with two identical tails, the surface shear viscosity rises rapidly with tail length, while the intermonolayer friction coefficient is less sensitive to the tail length. Interdigitation of lipid tails across the bilayer midsurface, as observed for lipids with two distinct tails, strongly enhances the intermonolayer friction coefficient, but hardly affects the surface shear viscosity. The simulation results are compared against the available experimental data. PMID:17468168

  5. Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid.

    PubMed

    Simon, S A; Disalvo, E A; Gawrisch, K; Borovyagin, V; Toone, E; Schiffman, S S; Needham, D; McIntosh, T J

    1994-06-01

    Tannic acid (TA) is a naturally occurring polyphenolic compound that aggregates membranes and neutral phosolipid vesicles and precipitates many proteins. This study analyzes TA binding to lipid membranes and the ensuing aggregation. The optical density of dispersions of phosphatidylcholine (PC) vesicles increased upon the addition of TA and electron micrographs showed that TA caused the vesicles to aggregate and form stacks of tightly packed disks. Solution calorimetry showed that TA bound to PC bilayers with a molar binding enthalpy of -8.3 kcal/mol and zeta potential measurements revealed that TA imparted a small negative charge to PC vesicles. Monolayer studies showed that TA bound to PC with a dissociation constant of 1.5 microM and reduced the dipole potential by up to 250 mV. Both the increase in optical density and decrease in dipole potential produced by TA could be reversed by the addition of polyvinylpyrrolidone, a compound that chelates TA by providing H-bond acceptor groups. NMR, micropipette aspiration, and x-ray diffraction experiments showed that TA incorporated into liquid crystalline PC membranes, increasing the area per lipid molecule and decreasing the bilayer thickness by 2 to 4%. 2H-NMR quadrupole splitting measurements also showed that TA associated with a PC molecule for times much less than 10(-4) s. In gel phase bilayers, TA caused the hydrocarbon chains from apposing monolayers to fully interdigitate. X-ray diffraction measurements of both gel and liquid crystalline dispersions showed that TA, at a critical concentration of about 1 mM, reduced the fluid spacing between adjacent bilayers by 8-10 A. These data place severe constraints on how TA can pack between adjacent bilayers and cause vesicles to adhere. We conclude that TA promotes vesicle aggregation by reducing the fluid spacing between bilayers by the formation of transient interbilayer bridges by inserting its digallic acid residues into the interfacial regions of adjacent bilayers

  6. Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function

    PubMed Central

    Andersson, Jakob; Köper, Ingo

    2016-01-01

    Solid supported bilayer lipid membranes are model systems to mimic natural cell membranes in order to understand structural and functional properties of such systems. The use of a model system allows for the use of a wide variety of analytical tools including atomic force microscopy, impedance spectroscopy, neutron reflectometry, and surface plasmon resonance spectroscopy. Among the large number of different types of model membranes polymer-supported and tethered lipid bilayers have been shown to be versatile and useful systems. Both systems consist of a lipid bilayer, which is de-coupled from an underlying support by a spacer cushion. Both systems will be reviewed, with an emphasis on the effect that the spacer moiety has on the bilayer properties. PMID:27249006

  7. Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function.

    PubMed

    Andersson, Jakob; Köper, Ingo

    2016-01-01

    Solid supported bilayer lipid membranes are model systems to mimic natural cell membranes in order to understand structural and functional properties of such systems. The use of a model system allows for the use of a wide variety of analytical tools including atomic force microscopy, impedance spectroscopy, neutron reflectometry, and surface plasmon resonance spectroscopy. Among the large number of different types of model membranes polymer-supported and tethered lipid bilayers have been shown to be versatile and useful systems. Both systems consist of a lipid bilayer, which is de-coupled from an underlying support by a spacer cushion. Both systems will be reviewed, with an emphasis on the effect that the spacer moiety has on the bilayer properties. PMID:27249006

  8. Interactions between lipid bilayers and inorganic material surfaces

    NASA Astrophysics Data System (ADS)

    Mager, Morgan Douglas

    Because of their unique biological and material properties, lipid bilayers have been extensively studied for use in biosensor and drug delivery applications. In the past, these systems have mostly taken the form of bulk solutions. More recently, researchers have integrated bilayers with chip-based architectures to take advantage of advanced optical, scanning probe and electronic characterization. These applications typically involve the creation of hybrid devices with inorganic and bilayer components, both of which affect the final device performance. In particular, the properties of supported lipid bilayers (SLBs) are known to depend on the substrate chemistry and topography as well as the lipid used. In spite of the large body of work involving these systems, there is still much that remains unknown about the formation and ultimate structure of the interface between these very different materials. One outstanding question in the study of SLBs is the role that the bilayer deposition method plays in determining the bilayer properties. In this work, we have developed a new method for forming and patterning lipid bilayers: bubble collapse deposition (BCD). This method is similar to an in situ version of Langmuir-Blodgett deposition, and offers unique possibilities for the fabrication of lipid-based devices. Briefly, a lipid monolayer is "inked" onto the surface of an air bubble. This bubble is then brought down on a solid support and the air is withdrawn. This withdrawal of air shrinks the bubble, which causes the monolayer to fold over on itself and redeposit on the surface as a bilayer. With BCD, we have demonstrated the first SLB formation on alumina using uncharged lipids. Using this system, we have measured a previously unobserved enhanced hydrodynamic coupling at the alumina surface. We have also used BCD to produce a hybrid lipid-gated chemical delivery device with potentially sub-cellular spatial resolution. Because of the unique material properties of the

  9. Photon correlation spectroscopy of bilayer lipid membranes.

    PubMed

    Crilly, J F; Earnshaw, J C

    1983-02-01

    Light scattering by thermal fluctuations on simple monoglyceride bilayer membranes has been used to investigate the viscoelastic properties of these structures. Spectroscopic analysis of these fluctuations (capillary waves) permits the nonperturbative measurement of the interfacial tension and a shear interfacial viscosity acting normal to the membrane plane. The methods were established by studies of solvent and nonsolvent bilayers of glycerol monooleate (GMO). Changes in the tension of GMO/n-decane membranes induced by altering the composition of the parent solution were detected and quantified. In a test of the reliability of the technique controlled variations of the viscosity of the aqueous bathing solution were accurately monitored. The technique was applied to solvent-free bilayers formed from dispersions of GMO in squalane. The lower tensions observed attested to the comparative absence of solvent in such bilayers. In contrast to the solvent case, the solvent-free membranes exhibited a significant transverse shear viscosity, indicative of the enhanced intermolecular interactions within the bilayer.

  10. Lipid Bilayer Membrane Perturbation by Embedded Nanopores: A Simulation Study.

    PubMed

    Garcia-Fandiño, Rebeca; Piñeiro, Ángel; Trick, Jemma L; Sansom, Mark S P

    2016-03-22

    A macromolecular nanopore inserted into a membrane may perturb the dynamic organization of the surrounding lipid bilayer. To better understand the nature of such perturbations, we have undertaken a systematic molecular dynamics simulation study of lipid bilayer structure and dynamics around three different classes of nanopore: a carbon nanotube, three related cyclic peptide nanotubes differing in the nature of their external surfaces, and a model of a β-barrel nanopore protein. Periodic spatial distributions of several lipid properties as a function of distance from the nanopore were observed. This was especially clear for the carbon nanotube system, for which the density of lipids, the bilayer thickness, the projection of lipid head-to-tail vectors onto the membrane plane, and lipid lateral diffusion coefficients exhibited undulatory behavior as a function of the distance from the surface of the channel. Overall, the differences in lipid behavior as a function of the nanopore structure reveal local adaptation of the bilayer structure and dynamics to different embedded nanopore structures. Both the local structure and dynamic behavior of lipids around membrane-embedded nanopores are sensitive to the geometry and nature of the outer surface of the macromolecule/molecular assembly forming the pore.

  11. Detection of supported lipid bilayers with carbon nanotube transistors

    NASA Astrophysics Data System (ADS)

    Zhou, Xinjian; Moran-Mirabal, Jose; Craighead, Harold; McEuen, Paul

    2005-03-01

    Supported lipid bilayers are important synthetic structures that can be used to mimic and study the properties and functions of cellular membranes, as well as to perform various bioassays which involve membrane bound receptors. The fusion of phospholipid vesicles and formation of a supported lipid bilayer can be detected in real time with high sensitivity by carbon nanotube field effect transistors which have been patterned on the same substrate. The properties of different vesicles, such as fusion rates and phospholipid composition can be distinguished by the conductance change of carbon nanotube field effect transistors. Fluorescence is used to verify the formation of a supported lipid bilayer, although the detection scheme is label-free. This demonstrates that electrical detection with carbon nanotubes can provide a powerful tool for study of lipid membranes.

  12. Actin Assembly at Model-Supported Lipid Bilayers

    PubMed Central

    Heath, George R.; Johnson, Benjamin R.G.; Olmsted, Peter D.; Connell, Simon D.; Evans, Stephen D.

    2013-01-01

    We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant—making this a practical approach for creating stable lipid-actin systems via self-assembly. PMID:24268147

  13. High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition.

    PubMed

    Watanabe, Rikiya; Soga, Naoki; Yamanaka, Tomoko; Noji, Hiroyuki

    2014-11-17

    We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

  14. High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition

    PubMed Central

    Watanabe, Rikiya; Soga, Naoki; Yamanaka, Tomoko; Noji, Hiroyuki

    2014-01-01

    We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications. PMID:25399694

  15. Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids

    PubMed Central

    Lind, Tania Kjellerup; Wacklin, Hanna; Schiller, Jürgen; Moulin, Martine; Haertlein, Michael; Pomorski, Thomas Günther; Cárdenas, Marité

    2015-01-01

    Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics. PMID:26658241

  16. Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids.

    PubMed

    Lind, Tania Kjellerup; Wacklin, Hanna; Schiller, Jürgen; Moulin, Martine; Haertlein, Michael; Pomorski, Thomas Günther; Cárdenas, Marité

    2015-01-01

    Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics. PMID:26658241

  17. Lipid bilayer modules as determinants of K+ channel gating.

    PubMed

    Syeda, Ruhma; Santos, Jose S; Montal, Mauricio

    2014-02-14

    The crystal structure of the sensorless pore module of a voltage-gated K(+) (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules.

  18. Binding Orientations and Lipid Interactions of Human Amylin at Zwitterionic and Anionic Lipid Bilayers

    PubMed Central

    Qian, Zhenyu; Jia, Yan; Wei, Guanghong

    2016-01-01

    Increasing evidence suggests that the interaction of human islet amyloid polypeptide (hIAPP) with lipids may facilitate hIAPP aggregation and cause the death of pancreatic islet β-cells. However, the detailed hIAPP-membrane interactions and the influences of lipid compositions are unclear. In this study, as a first step to understand the mechanism of membrane-mediated hIAPP aggregation, we investigate the binding behaviors of hIAPP monomer at zwitterionic palmitoyloleoyl-phosphatidylcholine (POPC) bilayer by performing atomistic molecular dynamics simulations. The results are compared with those of hIAPP at anionic palmitoyloleoyl-phosphatidylglycerol (POPG) bilayers. We find that the adsorption of hIAPP to POPC bilayer is mainly initiated from the C-terminal region and the peptide adopts a helical structure with multiple binding orientations, while the adsorption to POPG bilayer is mostly initiated from the N-terminal region and hIAPP displays one preferential binding orientation, with its hydrophobic residues exposed to water. hIAPP monomer inserts into POPC lipid bilayers more readily than into POPG bilayers. Peptide-lipid interaction analyses show that the different binding features of hIAPP at POPC and POPG bilayers are attributed to different magnitudes of electrostatic and hydrogen-bonding interactions with lipids. This study provides mechanistic insights into the different interaction behaviors of hIAPP with zwitterionic and anionic lipid bilayers. PMID:26649316

  19. Lipid bilayer microarray for parallel recording of transmembrane ion currents.

    PubMed

    Le Pioufle, Bruno; Suzuki, Hiroaki; Tabata, Kazuhito V; Noji, Hiroyuki; Takeuchi, Shoji

    2008-01-01

    This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

  20. Equilibrium Configurations of Lipid Bilayer Membranes and Carbon Nanostructures

    NASA Astrophysics Data System (ADS)

    Ivaïlo, M. Mladenov; Peter, A. Djondjorov; Mariana, Ts. Hadzhilazova; Vassil, M. Vassilev

    2013-02-01

    The present article concerns the continuum modelling of the mechanical behaviour and equilibrium shapes of two types of nano-scale objects: fluid lipid bilayer membranes and carbon nanostructures. A unified continuum model is used to handle four different case studies. Two of them consist in representing in analytic form cylindrical and axisymmetric equilibrium configurations of single-wall carbon nanotubes and fluid lipid bilayer membranes subjected to uniform hydrostatic pressure. The third one is concerned with determination of possible shapes of junctions between a single-wall carbon nanotube and a fiat graphene sheet or another single-wall carbon nanotube. The last one deals with the mechanical behaviour of closed fluid lipid bilayer membranes (vesicles) adhering onto a fiat homogeneous rigid substrate subjected to micro-injection and uniform hydrostatic pressure.

  1. Simulation of lipid bilayer self-assembly using all-atom lipid force fields.

    PubMed

    Skjevik, Åge A; Madej, Benjamin D; Dickson, Callum J; Lin, Charles; Teigen, Knut; Walker, Ross C; Gould, Ian R

    2016-04-21

    In this manuscript we expand significantly on our earlier communication by investigating the bilayer self-assembly of eight different types of phospholipids in unbiased molecular dynamics (MD) simulations using three widely used all-atom lipid force fields. Irrespective of the underlying force field, the lipids are shown to spontaneously form stable lamellar bilayer structures within 1 microsecond, the majority of which display properties in satisfactory agreement with the experimental data. The lipids self-assemble via the same general mechanism, though at formation rates that differ both between lipid types, force fields and even repeats on the same lipid/force field combination. In addition to zwitterionic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids, anionic phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids are represented. To our knowledge this is the first time bilayer self-assembly of phospholipids with negatively charged head groups is demonstrated in all-atom MD simulations.

  2. Irregular bilayer structure in vesicles prepared from Halobacterium cutirubrum lipids

    NASA Technical Reports Server (NTRS)

    Lanyi, J. K.

    1974-01-01

    Fluorescent probes were used to study the structure of the cell envelope of Halobacterium cutirubrum, and, in particular, to explore the effect of the heterogeneity of the lipids in this organism on the structure of the bilayers. The fluorescence polarization of perylene was followed in vesicles of unfractionated lipids and polar lipids as a function of temperature in 3.4 M solutions of NaCl, NaNO3, and KSCN, and it was found that vesicles of unfractionated lipids were more perturbed by chaotropic agents than polar lipids. The dependence of the relaxation times of perylene on temperature was studied in cell envelopes and in vesicles prepared from polar lipids, unfractionated lipids, and mixtures of polar and neutral lipids.

  3. Structural studies of polymer-cushioned lipid bilayers.

    PubMed

    Majewski, J; Wong, J Y; Park, C K; Seitz, M; Israelachvili, J N; Smith, G S

    1998-11-01

    The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.

  4. Solvent-free simulations of heterogeneous lipid bilayers

    NASA Astrophysics Data System (ADS)

    Brannigan, Grace H.

    Two molecular implicit solvent models for fluid lipid bilayers are presented. The first represents lipids by rigid, asymmetric, soft spherocylinders. The three parameter potential between pairs of lipids gives rise to micelles, fluid bilayers, and gel-like bilayers. Fluid bilayers have compressibility moduli in agreement with experimental systems but display bending moduli at least three times larger than typical biological membranes without cholesterol. The second three parameter model represents lipids by flexible chains of beads; the hydrophobic effect is mimicked through a soft pair potential localized at the interface between hydrophobic and hydrophilic beads. Fluid bilayers composed of these molecules have both compressibility moduli and elastic moduli in agreement with experimental systems, as well as realistic interfacial tensions and stress profiles. Monte Carlo simulations are used to demonstrate self-assembly for both models. Phase behavior is studied for bilayers composed of the rigid model. Regions of solid, "hexatic", and fluid bilayer behavior are established by identification of phase boundaries. The main melting transition is found to be density driven; the melting temperature scales inversely with lipid length since thermal expansion increases with lipid aspect ratio. A plausible sub-transition is identified for longer molecules. The dependence of membrane elasticity on bilayer thickness is obtained by adjusting the length of the rigid molecules at constant temperature and surface tension. The bending modulus scales with the square of the membrane thickness, as expected, but the proportionality constant is an order of magnitude smaller than expected using continuum elastic theories or measured by experiments. The proportionality constant is found to be non-monotonic for bilayers composed of multiple lipid species; the thinnest membranes are not the most flexible. This is shown to be quantitatively consistent with the random quadratic mixing

  5. Modifiers of the Dipole Potential of Lipid Bilayers

    PubMed Central

    Efimova, S. S.; Ostroumova, O. S.

    2015-01-01

    This paper assesses the magnitude of change in the dipole potential (φd) of membranes caused by the adsorption of modifiers on lipid bilayers of various compositions. We tested flavonoids, muscle relaxants, thyroid hormones, and xanthene and styrylpyridinium dyes in order to assess their dipole-modifying properties. A quantitative description of the modifying action of flavonoids, muscle relaxants, thyroid hormones, and xanthene dyes is shown as the ratio of the maximum change in the bilayer dipole potential upon saturation and the absolute φd value of the unmodified membrane. The slopes of the linear relationship between the increase in the dipole potential of phospholipid bilayers and the concentration of styrylpyridinium dyes in membrane-bathing solutions were found. We described the relationships between the change in φd and the chemical structure of modifiers, as well as the charge and spontaneous curvature of lipid monolayers. PMID:26798493

  6. [Lipid oxidation in bilayer lipid membranes linked with the reaction of oxidation of NAD.H by atmospheric oxygen].

    PubMed

    Shchipumov, Iu A; Sokolov, V S; Iaguzhinskiĭ, L S; Boguslavskiĭ, L I

    1976-01-01

    It is shown that along with NAD.H oxidation with air oxygen peroxide oxidation of lipids forming the membrane takes place in bilayer lipid membranes modified with ubiquinone. During nicotin amide oxidation proton absorption takes place. Peroxide oxidation of lipids results in the liberation of H+ ions, which in its turn brings about the formation of protone-deficient or enriched (against aqueous solution) layers adjacent to the membrane. The potential value on the membrane is shown to depend on nicotine amide and oxygen concentration, on ubiquinone presence and lipid composition of the membrane. It has been also indicated that the transmembrane potential difference is initiated with a sharp change of aqueous solution pH by 0.05--0.4 units. PMID:178383

  7. Anthrax toxin-induced rupture of artificial lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

    2013-08-01

    We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

  8. Supported lipid bilayer microarrays created by non-contact printing.

    PubMed

    Kaufmann, Stefan; Sobek, Jens; Textor, Marcus; Reimhult, Erik

    2011-07-21

    Arrays of supported lipid bilayers (SLBs) provide great potential for future drug development and multiplexed biological research, but are difficult to prepare due to the sensitivity of both the lipid and protein structural arrangement to air exposure. A novel way to produce arrays of SLBs is presented based on non-contact dispensing of vesicles to a substrate through a thin surface confined water film. The approach presents many degrees of freedom since it is not limited to a specific substrate, lipid composition, linker or controlled environment. The method allows adjustment of spot size (180-360 μm) by repeated dispensing as well as control over the composition of the spots and subsequent analytes. SLB formation by vesicle adsorption and rupture allows for incorporation of membrane proteins through pre-formed proteoliposomes. Dispensing through a dip-and-rinse water film avoids contamination, disruptive drying and the need for complex buffer compositions. Furthermore, no humidity control is necessary which simplifies the production step and prolongs the life-time of the spotting system. We characterize the method with respect to control over spot size, bilayer mobility and the formation process as well as demonstrate the possibility to fuse bilayer spots with subsequently added vesicles. Since complex lipid compositions and multiple spotting nozzles can be used, this novel technique is expected to be a promising platform for future applications, e.g. patterning to monitor peptide/protein-lipid interactions, for glycomics using glycolipids or lipopolysaccharides, and to study mixing of spatially confined lipid membranes.

  9. Protein-induced bilayer perturbations: Lipid ordering and hydrophobic coupling.

    PubMed

    Petersen, Frederic N R; Laursen, Ib; Bohr, Henrik; Nielsen, Claus Hélix

    2009-10-01

    The host lipid bilayer is increasingly being recognized as an important non-specific regulator of membrane protein function. Despite considerable progress the interplay between hydrophobic coupling and lipid ordering is still elusive. We use electron spin resonance (ESR) to study the interaction between the model protein gramicidin and lipid bilayers of varying thickness. The free energy of the interaction is up to -6kJ/mol; thus not strongly favored over lipid-lipid interactions. Incorporation of gramicidin results in increased order parameters with increased protein concentration and hydrophobic mismatch. Our findings also show that at high protein:lipid ratios the lipids are motionally restricted but not completely immobilized. Both exchange on and off rate values for the lipid<-->gramicidin interaction are lowest at optimal hydrophobic matching. Hydrophobic mismatch of few A results in up to 10-fold increased exchange rates as compared to the 'optimal' match situation pointing to the regulatory role of hydrophobic coupling in lipid-protein interactions.

  10. Protein-induced bilayer perturbations: Lipid ordering and hydrophobic coupling

    SciTech Connect

    Petersen, Frederic N.R.; Laursen, Ib; Bohr, Henrik; Nielsen, Claus Helix

    2009-10-02

    The host lipid bilayer is increasingly being recognized as an important non-specific regulator of membrane protein function. Despite considerable progress the interplay between hydrophobic coupling and lipid ordering is still elusive. We use electron spin resonance (ESR) to study the interaction between the model protein gramicidin and lipid bilayers of varying thickness. The free energy of the interaction is up to -6 kJ/mol; thus not strongly favored over lipid-lipid interactions. Incorporation of gramicidin results in increased order parameters with increased protein concentration and hydrophobic mismatch. Our findings also show that at high protein:lipid ratios the lipids are motionally restricted but not completely immobilized. Both exchange on and off rate values for the lipid {r_reversible} gramicidin interaction are lowest at optimal hydrophobic matching. Hydrophobic mismatch of few A results in up to 10-fold increased exchange rates as compared to the 'optimal' match situation pointing to the regulatory role of hydrophobic coupling in lipid-protein interactions.

  11. The assembly and use of tethered bilayer lipid membranes (tBLMs).

    PubMed

    Cranfield, Charles; Carne, Sonia; Martinac, Boris; Cornell, Bruce

    2015-01-01

    Because they are firmly held in place, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cornell et al. Nature 387(6633): 580-583, 1997). Here we describe the procedures required to assemble and test tethered lipid bilayers that can incorporate various lipid species, peptides, and ion channel proteins. PMID:25331126

  12. Gravimetric antigen detection utilizing antibody-modified lipid bilayers.

    PubMed

    Larsson, Charlotte; Bramfeldt, Hanna; Wingren, Christer; Borrebaeck, Carl; Höök, Fredrik

    2005-10-01

    Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO2 have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni2+-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing GM1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.

  13. Effect of Sodium Chloride on a Lipid Bilayer

    PubMed Central

    Böckmann, Rainer A.; Hac, Agnieszka; Heimburg, Thomas; Grubmüller, Helmut

    2003-01-01

    Electrostatic interactions govern structural and dynamical properties of membranes and can vary considerably with the composition of the aqueous buffer. We studied the influence of sodium chloride on a pure POPC lipid bilayer by fluorescence correlation spectroscopy experiments and molecular dynamics simulations. Increasing sodium chloride concentration was found to decrease the self-diffusion of POPC lipids within the bilayer. Self-diffusion coefficients calculated from the 100 ns simulations agree with those measured on a millisecond timescale, suggesting that most of the relaxation processes relevant for lipid diffusion are faster than the simulation timescale. As the dominant effect, the molecular dynamics simulations revealed a tight binding of sodium ions to the carbonyl oxygens of on average three lipids leading to larger complexes with reduced mobility. Additionally, the bilayer thickens by ∼2 Å, which increases the order parameter of the fatty acyl chains. Sodium binding alters the electrostatic potential, which is largely compensated by a changed polarization of the aqueous medium and a lipid dipole reorientation. PMID:12944279

  14. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers

    PubMed Central

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-01-01

    Summary Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction. PMID:20547132

  15. Collective lipid bilayer dynamics excited by surface acoustic waves.

    PubMed

    Reusch, T; Schülein, F J R; Nicolas, J D; Osterhoff, M; Beerlink, A; Krenner, H J; Müller, M; Wixforth, A; Salditt, T

    2014-09-12

    We use standing surface acoustic waves to induce coherent phonons in model lipid multilayers deposited on a piezoelectric surface. Probing the structure by phase-controlled stroboscopic x-ray pulses we find that the internal lipid bilayer electron density profile oscillates in response to the externally driven motion of the lipid film. The structural response to the well-controlled motion is a strong indication that bilayer structure and membrane fluctuations are intrinsically coupled, even though these structural changes are averaged out in equilibrium and time integrating measurements. Here the effects are revealed by a timing scheme with temporal resolution on the picosecond scale in combination with the sub-nm spatial resolution, enabled by high brilliance synchrotron x-ray reflectivity.

  16. How antimicrobial peptides disrupt lipid bilayers?

    NASA Astrophysics Data System (ADS)

    Sengupta, Durba

    2011-03-01

    The molecular basis for the activity of cyclic and linear antimicrobial peptides is analysed. We performed multi-scale molecular dynamics simulations and biophysical measurements to probe the interaction of antimicrobial peptides with model membranes. Two linear antimicrobial peptides, magainin and melittin and a cyclic one, BPC194 have been studied. We test different models to determine the generic and specific forces that lead to bilayer disruption. We probe whether interfacial stress or local membrane perturbation is more likely to lead to the porated state. We further analyse the reasons that determine specificity and increase of activity in antimicrobial peptides. The results provide detailed insight in the mode of action of antimicrobial peptides.

  17. Dynamics and instabilities of lipid bilayer membrane shapes.

    PubMed

    Shi, Zheng; Baumgart, Tobias

    2014-06-01

    Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that describe the fluctuations (i.e. undulations) of bilayer shapes as well as their local coupling with lipid or protein density variation. The coupling between local membrane curvature and lipid density gives rise to a 'slipping mode' in addition to the conventional 'bending mode' for damping the membrane fluctuation. This leads to a number of interesting experimental phenomena regarding bilayer shape dynamics. More importantly, curvature-inducing proteins can couple with membrane shape and eventually render the membrane unstable. A criterion for membrane shape instability is derived from a linear stability analysis. The instability criterion reemphasizes the importance of membrane tension in regulating the stability and dynamics of membrane geometry. Recent progresses in understanding the role of membrane tension in regulating dynamical cellular processes are also reviewed. Protein density is emphasized as a key factor in regulating membrane shape transitions: a threshold density of curvature coupling proteins is required for inducing membrane morphology transitions. PMID:24529968

  18. Dynamics and instabilities of lipid bilayer membrane shapes

    PubMed Central

    Shi, Zheng; Baumgart, Tobias

    2014-01-01

    Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that describe the fluctuations (i.e. undulations) of bilayer shapes as well as their local coupling with lipid or protein density variation. The coupling between local membrane curvature and lipid density gives rise to a ‘slipping mode’ in addition to the conventional ‘bending mode’ for damping the membrane fluctuation. This leads to a number of interesting experimental phenomena regarding bilayer shape dynamics. More importantly, curvature-inducing proteins can couple with membrane shape and eventually render the membrane unstable. A criterion for membrane shape instability is derived from a linear stability analysis. The instability criterion reemphasizes the importance of membrane tension in regulating the stability and dynamics of membrane geometry. Recent progresses in understanding the role of membrane tension in regulating dynamical cellular processes are also reviewed. Protein density is emphasized as a key factor in regulating membrane shape transitions: a threshold density of curvature coupling proteins is required for inducing membrane morphology transitions. PMID:24529968

  19. Naratriptan aggregation in lipid bilayers: perspectives from molecular dynamics simulations.

    PubMed

    Wood, Irene; Pickholz, Mónica

    2016-09-01

    In order to understand the interaction between naratriptan and a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC), we carried out molecular dynamics simulations. The simulations were performed considering neutral and protonated ionization states, starting from different initial conditions. At physiological pH, the protonated state of naratriptan is predominant. It is expected that neutral compounds could have larger membrane partition than charged compounds. However, for the specific case of triptans, it is difficult to study neutral species in membranes experimentally, making computer simulations an interesting tool. When the naratriptan molecules were originally placed in water, they partitioned between the bilayer/water interface and water phase, as has been described for similar compounds. From this condition, the drugs displayed low access to the hydrophobic environment, with no significant effects on bilayer organization. The molecules anchored in the interface, due mainly to the barrier function of the polar and oriented lipid heads. On the other hand, when placed inside the bilayer, both neutral and protonated naratriptan showed self-aggregation in the lipid tail environment. In particular, the protonated species exhibited a pore-like structure, dragging water through this environment. Graphical Abstract Different behaviour of Naratriptan and Sumatriptan, when the drugs were originally placed in the lipid core. PMID:27558798

  20. Penetration of Cell Membranes and Synthetic Lipid Bilayers by Nanoprobes

    PubMed Central

    Angle, Matthew R.; Wang, Andrew; Thomas, Aman; Schaefer, Andreas T.; Melosh, Nicholas A.

    2014-01-01

    Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50–500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe's sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes. PMID:25418094

  1. Manipulating lipid bilayer material properties using biologically active amphipathic molecules

    NASA Astrophysics Data System (ADS)

    Ashrafuzzaman, Md; Lampson, M. A.; Greathouse, D. V.; Koeppe, R. E., II; Andersen, O. S.

    2006-07-01

    Lipid bilayers are elastic bodies with properties that can be manipulated/controlled by the adsorption of amphipathic molecules. The resulting changes in bilayer elasticity have been shown to regulate integral membrane protein function. To further understand the amphiphile-induced modulation of bilayer material properties (thickness, intrinsic monolayer curvature and elastic moduli), we examined how an enantiomeric pair of viral anti-fusion peptides (AFPs)—Z-Gly-D-Phe and Z-Gly-Phe, where Z denotes a benzyloxycarbonyl group, as well as Z-Phe-Tyr and Z-D-Phe-Phe-Gly—alters the function of enantiomeric pairs of gramicidin channels of different lengths in planar bilayers. For both short and long channels, the channel lifetimes and appearance frequencies increase as linear functions of the aqueous AFP concentration, with no apparent effect on the single-channel conductance. These changes in channel function do not depend on the chirality of the channels or the AFPs. At pH 7.0, the relative changes in channel lifetimes do not vary when the channel length is varied, indicating that these compounds exert their effects primarily by causing a positive-going change in the intrinsic monolayer curvature. At pH 4.0, the AFPs are more potent than at pH 7.0 and have greater effects on the shorter channels, indicating that these compounds now change the bilayer elastic moduli. When AFPs of different anti-fusion potencies are compared, the rank order of the anti-fusion activity and the channel-modifying activity is similar, but the relative changes in anti-fusion potency are larger than the changes in channel-modifying activity. We conclude that gramicidin channels are useful as molecular force transducers to probe the influence of small amphiphiles upon lipid bilayer material properties.

  2. Nonadditive Compositional Curvature Energetics of Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Sodt, A. J.; Venable, R. M.; Lyman, E.; Pastor, R. W.

    2016-09-01

    The unique properties of the individual lipids that compose biological membranes together determine the energetics of the surface. The energetics of the surface, in turn, govern the formation of membrane structures and membrane reshaping processes, and thus they will underlie cellular-scale models of viral fusion, vesicle-dependent transport, and lateral organization relevant to signaling. The spontaneous curvature, to the best of our knowledge, is always assumed to be additive. We describe observations from simulations of unexpected nonadditive compositional curvature energetics of two lipids essential to the plasma membrane: sphingomyelin and cholesterol. A model is developed that connects molecular interactions to curvature stress, and which explains the role of local composition. Cholesterol is shown to lower the number of effective Kuhn segments of saturated acyl chains, reducing lateral pressure below the neutral surface of bending and favoring positive curvature. The effect is not observed for unsaturated (flexible) acyl chains. Likewise, hydrogen bonding between sphingomyelin lipids leads to positive curvature, but only at sufficient concentration, below which the lipid prefers negative curvature.

  3. Dynamic and mechanical properties of supported lipid bilayers

    NASA Astrophysics Data System (ADS)

    Wu, Hsing-Lun; Tsao, Heng-Kwong; Sheng, Yu-Jane

    2016-04-01

    Supported lipid bilayers (SLBs) offer an excellent model system for investigating the physico-chemical properties of the cell membrane. In this work, dynamic and mechanical properties of SLBs are explored by dissipative particle dynamics simulations for lipids with different architectures (chain length, kink, and asymmetry associated with lipid tails). It is found that the lateral diffusivity (Dx) and flip-flop rate (FF) grow with increasing temperature in both gel and liquid phases and can be described by an Arrhenius-like expression. Three regimes can be clearly identified for symmetric and asymmetric saturated lipids but only two regimes are observed for kinked lipids. Both Dx and FF grow with decreasing tail length and increasing number of kinks. The stretching (KA) and apparent bending (KB) moduli exhibit concave upward curves with temperature and the minima are attained at Tm. In general, the minima of KA and KB decrease with the chain length and increase with number of kinks. The typical relation among the bending modulus, area stretching modulus, and bilayer thickness is still followed, KB = βKAh2 and β is much smaller in the gel phase. The dynamic and mechanical properties of lipids with asymmetric tails are found to situate between their symmetric counterparts.

  4. Melittin-induced cholesterol reorganization in lipid bilayer membranes

    DOE PAGES

    Qian, Shuo; Heller, William T.

    2015-06-12

    The peptide melittin, a 26 amino acid, cationic peptide from honey bee (Apis mellifera) venom, disrupts lipid bilayer membranes in a concentration-dependent manner. Rather than interacting with a specific receptor, the peptide interacts directly with the lipid matrix of the membrane in a manner dependent on the lipid composition. Here, a small-angle neutron scattering study of the interaction of melittin with lipid bilayers made of mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) is presented. Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations below those thatmore » cause pores to form. In addition to disrupting the in-plane organization of Chol, melittin produces vesicles having inner and outer leaflet compositions that depend on the lipid–Chol molar ratio and on the peptide concentration. The changes seen at high cholesterol and low peptide concentration are similar to those produced by alamethicin (Qian, S. et al., J. Phys. Chem. B 2014, 118, 11200–11208), which points to an underlying physical mechanism driving the redistribution of Chol, but melittin displays an additional effect not seen with alamethicin. Furthermore, a model for how the peptide drives the redistribution of Chol is proposed. The results suggest that redistribution of the lipids in a target cell membrane by membrane active peptides takes places as a prelude to the lysis of the cell.« less

  5. Melittin-induced cholesterol reorganization in lipid bilayer membranes

    SciTech Connect

    Qian, Shuo; Heller, William T.

    2015-06-12

    The peptide melittin, a 26 amino acid, cationic peptide from honey bee (Apis mellifera) venom, disrupts lipid bilayer membranes in a concentration-dependent manner. Rather than interacting with a specific receptor, the peptide interacts directly with the lipid matrix of the membrane in a manner dependent on the lipid composition. Here, a small-angle neutron scattering study of the interaction of melittin with lipid bilayers made of mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) is presented. Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations below those that cause pores to form. In addition to disrupting the in-plane organization of Chol, melittin produces vesicles having inner and outer leaflet compositions that depend on the lipid–Chol molar ratio and on the peptide concentration. The changes seen at high cholesterol and low peptide concentration are similar to those produced by alamethicin (Qian, S. et al., J. Phys. Chem. B 2014, 118, 11200–11208), which points to an underlying physical mechanism driving the redistribution of Chol, but melittin displays an additional effect not seen with alamethicin. Furthermore, a model for how the peptide drives the redistribution of Chol is proposed. The results suggest that redistribution of the lipids in a target cell membrane by membrane active peptides takes places as a prelude to the lysis of the cell.

  6. Dynamic and mechanical properties of supported lipid bilayers.

    PubMed

    Wu, Hsing-Lun; Tsao, Heng-Kwong; Sheng, Yu-Jane

    2016-04-21

    Supported lipid bilayers (SLBs) offer an excellent model system for investigating the physico-chemical properties of the cell membrane. In this work, dynamic and mechanical properties of SLBs are explored by dissipative particle dynamics simulations for lipids with different architectures (chain length, kink, and asymmetry associated with lipid tails). It is found that the lateral diffusivity (Dx) and flip-flop rate (FF) grow with increasing temperature in both gel and liquid phases and can be described by an Arrhenius-like expression. Three regimes can be clearly identified for symmetric and asymmetric saturated lipids but only two regimes are observed for kinked lipids. Both Dx and FF grow with decreasing tail length and increasing number of kinks. The stretching (KA) and apparent bending (KB) moduli exhibit concave upward curves with temperature and the minima are attained at Tm. In general, the minima of KA and KB decrease with the chain length and increase with number of kinks. The typical relation among the bending modulus, area stretching modulus, and bilayer thickness is still followed, KB = βKAh(2) and β is much smaller in the gel phase. The dynamic and mechanical properties of lipids with asymmetric tails are found to situate between their symmetric counterparts. PMID:27389237

  7. Molecular Dynamics Simulations of Hydrophilic Pores in Lipid Bilayers

    PubMed Central

    Leontiadou, Hari; Mark, Alan E.; Marrink, Siewert J.

    2004-01-01

    Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of ∼38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be ∼3 × 10−11 N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores. PMID:15041656

  8. Mechanical properties of lipid bilayers and regulation of mechanosensitive function

    PubMed Central

    Balleza, Daniel

    2012-01-01

    Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed. PMID:22790280

  9. Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

    PubMed Central

    Crites, Travis J.; Maddox, Michael; Padhan, Kartika; Muller, James; Eigsti, Calvin; Varma, Rajat

    2015-01-01

    Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology. PMID:26331983

  10. Electrochemical characterization of bilayer lipid membrane-semiconductor junctions

    SciTech Connect

    Zhao, Xiao Kang; Baral, S.; Fendler, J.H. )

    1990-03-08

    Three different systems of glyceryl monooleate (GMO), bilayer lipid membrane (BLM) supported semiconductor particles have been prepared and characterized. A single composition of particulate semiconductor deposited only on one side of the BLM constituted system A, two different compositions of particulate semiconductors sequentially deposited on the same side of the BLM represented system B, and two different compositions of particulate semiconductors deposited on the opposite sides of the BLM made up system C.

  11. Multivalent Ligand-Receptor Binding on Supported Lipid Bilayers

    PubMed Central

    Jung, Hyunsook; Robison, Aaron D.; Cremer, Paul S.

    2009-01-01

    Fluid supported lipid bilayers provide an excellent platform for studying multivalent protein-ligand interactions because the two-dimensional fluidity of the membrane allows for lateral rearrangement of ligands in order to optimize binding. Our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy (TIRFM) to obtain equilibrium dissociation constant (KD) data for these systems. This high throughput, on-chip approach provides highly accurate thermodynamic information about multivalent binding events while requiring only very small sample volumes. Herein, we review some of the most salient findings from these studies. In particular, increasing ligand density on the membrane surface can provide a modest enhancement or attenuation of ligand-receptor binding depending upon whether the surface ligands interact strongly with each other. Such effects, however, lead to little more than one order of magnitude change in the apparent KD values. On the other hand, the lipophilicity and presentation of lipid bilayer-conjugated ligands can have a much greater impact. Indeed, changing the way a particular ligand is conjugated to the membrane can alter the apparent KD value by at least three orders of magnitude. Such a result speaks strongly to the role of ligand availability for multivalent ligand-receptor binding. PMID:19508894

  12. The interaction of bee melittin with lipid bilayer membranes.

    PubMed

    Dawson, C R; Drake, A F; Helliwell, J; Hider, R C

    1978-06-16

    The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.

  13. Supported lipid bilayer nanosystems: stabilization by undulatory-protrusion forces and destabilization by lipid bridging.

    PubMed

    Savarala, Sushma; Monson, Frederick; Ilies, Marc A; Wunder, Stephanie L

    2011-05-17

    Control of the stabilization/destabilization of supported lipid bilayers (SLBs) on nanoparticles is important for promotion of their organized assembly and for their use as delivery vehicles. At the same time, understanding the mechanism of these processes can yield insight into nanoparticle-cell interactions and nanoparticle toxicity. In this study, the suspension/precipitation process of zwitterionic lipid/SiO(2) nanosystems was analyzed as a function of ionic strength and as a function of the ratio of lipid/SiO(2) surface areas, at pH = 7.6. Salt is necessary to induce supported lipid bilayer (SLB) formation for zwitterionic lipids on silica (SiO(2)) (Seantier, B.; Kasemo, B., Influence of Mono- and Divalent Ions on the Formation of Supported Phospholipid Bilayers via Vesicle Adsorption. Langmuir 2009, 25 (10), 5767-5772). However, for zwitterionic SLBs on SiO(2) nanoparticles, addition of salt can cause precipitation of the SLBs, due to electrostatic shielding by both the lipid and the salt and to the suppression of thermal undulation/protrusion repulsive forces for lipids on solid surfaces. At ionic strengths that cause precipitation of SLBs, it was found that addition of excess SUVs, at ratios where there were equal populations of SUVs and SLBs, restored the undulation/protrusion repulsive forces and restabilized the suspensions. We suggest that SUVs separate SLBs in the suspension, as observed by TEM, and that SLB-SLB interactions are replaced by SLB-SUV interactions. Decreasing the relative amount of lipid, to the extent that there was less lipid available than the amount required for complete bilayer coverage of the SiO(2), resulted in precipitation of the nanosystem by a process of nanoparticle lipid bridging. For this case, we postulate a process in which lipid bilayer patches on one nanoparticle collide with bare silica patches on another SiO(2) nanoparticle, forming a single bilayer bridge between them. TEM data confirmed these findings, thus

  14. Lipid domains control myelin basic protein adsorption and membrane interactions between model myelin lipid bilayers.

    PubMed

    Lee, Dong Woog; Banquy, Xavier; Kristiansen, Kai; Kaufman, Yair; Boggs, Joan M; Israelachvili, Jacob N

    2014-02-25

    The surface forces apparatus and atomic force microscope were used to study the effects of lipid composition and concentrations of myelin basic protein (MBP) on the structure of model lipid bilayers, as well as the interaction forces and adhesion between them. The lipid bilayers had a lipid composition characteristic of the cytoplasmic leaflets of myelin from "normal" (healthy) and "disease-like" [experimental allergic encephalomyelitis (EAE)] animals. They showed significant differences in the adsorption mechanism of MBP. MBP adsorbs on normal bilayers to form a compact film (3-4 nm) with strong intermembrane adhesion (∼0.36 mJ/m(2)), in contrast to its formation of thicker (7-8 nm) swelled films with weaker intermembrane adhesion (∼0.13 mJ/m(2)) on EAE bilayers. MBP preferentially adsorbs to liquid-disordered submicron domains within the lipid membranes, attributed to hydrophobic attractions. These results show a direct connection between the lipid composition of membranes and membrane-protein adsorption mechanisms that affects intermembrane spacing and adhesion and has direct implications for demyelinating diseases.

  15. A comparison of DMPC- and DLPE-based lipid bilayers.

    PubMed Central

    Damodaran, K V; Merz, K M

    1994-01-01

    A 250 ps molecular dynamics simulation of the dimyristoylphosphatidylcholine (DMPC)-based lipid bilayer, including explicit water molecules, is reported. The solvent environment of the head groups and other structural properties of the bilayer have been analyzed and compared with experimental results as well as our previous simulation of the dilauroylphosphatidylethanolamine (DLPE)-based bilayer. From this comparison we find that the solvent structure around the DMPC head group (clathrate shell) is significantly different than that around the DLPE head group (typical hydrogen bonding interactions). We have modeled the probable relationship between the different solvent environments around the R-N(CH3)3+ (DMPC) and R-NH3+ (DLPE) head groups and the different interlammelar distances in these systems by performing potential of mean force (PMF) simulations on two N(CH3)4+ and NH4+ ions in water. From the PMF simulations it appears that the differences in the hydration of the DMPC and DLPE head groups is not responsible for the differences in the hydration force observed for these systems. We also find that the orientational polarization of DLPE and DMPC is similar, which suggests that solvent polarization is not responsible for the differences in the hydration repulsion behavior observed in these systems. We also examined the order parameters for DMPC and found them to be in reasonable agreement with experiment. Given the different characteristics of the DLPE and DMPC head groups, we suggest an explanation of the differences in the interlammellar spacings of bilayers composed of these like-charged lipids. From our DLPE simulations we find that the R-NH3+ head groups can interact with the nonesterified oxygens of the phosphate group in an intraleaflet or an interleaflet manner. For the latter a "cross link" between two leaflets can be formed, which causes a stabilization of the interlamellar spacings at fairly short distances. Moreover, due to the strong intraleaflet

  16. Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

    PubMed Central

    Koldsø, Heidi; Shorthouse, David; Hélie, Jean; Sansom, Mark S. P.

    2014-01-01

    Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins. PMID:25340788

  17. Statistical mechanics and dynamics of two supported stacked lipid bilayers.

    PubMed

    Manghi, Manoel; Destainville, Nicolas

    2010-03-16

    The statistical physics and dynamics of double supported bilayers are studied theoretically. The main goal in designing double supported lipid bilayers is to obtain model systems of biomembranes: the upper bilayer is meant to be almost freely floating, the substrate being screened by the lower bilayer. The fluctuation-induced repulsion between membranes and between the lower membrane and the wall are explicitly taken into account using a Gaussian variational approach. It is shown that the variational parameters, the "effective" adsorption strength, and the average distance to the substrate, depend strongly on temperature and membrane elastic moduli, the bending rigidity, and the microscopic surface tension, which is a signature of the crucial role played by membrane fluctuations. The range of stability of these supported membranes is studied, showing a complex dependence on bare adsorption strengths. In particular, the experimental conditions of having an upper membrane slightly perturbed by the lower one and still bound to the surface are found. Included in the theoretical calculation of the damping rates associated with membrane normal modes are hydrodynamic friction by the wall and hydrodynamic interactions between both membranes. PMID:20000797

  18. Enzyme modification of platinum microelectrodes for detection of cholesterol in vesicle lipid bilayer membranes.

    PubMed

    Devadoss, Anando; Palencsár, M Simona; Jiang, Dechen; Honkonen, Michael L; Burgess, James D

    2005-11-15

    Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content. PMID:16285691

  19. The Interaction of Melittin with Dimyristoyl Phosphatidylcholine-Dimyristoyl Phosphatidylserine Lipid Bilayer Membranes

    DOE PAGES

    Rai, Durgesh K.; Qian, Shuo; Heller, William T.

    2016-08-13

    We report that membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine andmore » phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L = 1/500, but membrane thinning results when P/L = 1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. Lastly, the results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes.« less

  20. Modeling the Elastic Properties of Lipid Bilayer Membranes

    NASA Astrophysics Data System (ADS)

    Barry, Edward; Gibaud, Thomas; Zakhary, Mark; Dogic, Zvonimir

    2011-03-01

    Model membranes such as lipid bilayers have been indispensable tools for our understanding of the elastic properties of biological membranes. In this talk, I will introduce a colloidal model for membranes and demonstrate that the physical properties of these colloidal membranes are identical to lipid bilayers. The model system is unique in that the constituent molecules are homogenous and non-amphiphilic, yet their self-assembly into membranes and other hierarchical assemblages, such as a lamellar type phases and chiral ribbons, proceeds spontaneously in solution. Owing to the large size of the constituent molecules, individual molecules can be directly visualized and simultaneous observations at the continuum and molecular lengthscales are used to characterize the behavior of model membranes with unprecedented detail. Moreover, once assembled in solution, molecular interactions can be controlled in situ. In particular, the strength of chiral interactions can be varied, leading to fascinating transitions in behavior that resembles the formation of starfish vesicles. These observations point towards the important role of line tension, and have potential implications for phase separated lipid mixtures or lipid rafts.

  1. Electrowetting on dielectric-based microfluidics for integrated lipid bilayer formation and measurement

    NASA Astrophysics Data System (ADS)

    Poulos, Jason L.; Nelson, Wyatt C.; Jeon, Tae-Joon; Kim, Chang-Jin ``Cj''; Schmidt, Jacob J.

    2009-07-01

    We present a microfluidic platform for the formation and electrical measurement of lipid bilayer membranes. Using electrowetting on dielectric (EWOD), two or more aqueous droplets surrounded by a lipid-containing organic phase were manipulated into contact to form a lipid bilayer at their interface. Thin-film Ag/AgCl electrodes integrated into the device enabled electrical measurement of membrane formation and the incorporation of gramicidin channels of two bilayers in parallel.

  2. Formation and finite element analysis of tethered bilayer lipid structures.

    PubMed

    Kwak, Kwang Joo; Valincius, Gintaras; Liao, Wei-Ching; Hu, Xin; Wen, Xuejin; Lee, Andrew; Yu, Bo; Vanderah, David J; Lu, Wu; Lee, L James

    2010-12-01

    Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/β-mercaptoethanol (βME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI

  3. Formation and finite element analysis of tethered bilayer lipid structures.

    PubMed

    Kwak, Kwang Joo; Valincius, Gintaras; Liao, Wei-Ching; Hu, Xin; Wen, Xuejin; Lee, Andrew; Yu, Bo; Vanderah, David J; Lu, Wu; Lee, L James

    2010-12-01

    Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/β-mercaptoethanol (βME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI

  4. kT-Scale interactions between supported lipid bilayers.

    PubMed

    Everett, W Neil; Bevan, Michael A

    2014-01-14

    We use total internal reflection microscopy (TIRM) and confocal laser scanning microscopy (CSLM) to study supported lipid bilayer (SLB)-modified silica colloids with various SLB compositions (e.g., PEGylated vs. non-PEGylated) that control colloidal and bilayer stability. Measured and predicted potentials accurately capture stable configurations. For unstable conditions when SLBs adhere, fuse, or spread between surfaces, SLB structures are connected to effective potentials as well as time-dependent behavior. In all cases, directly measured and inferred interactions are well described by steric interactions between PEG brushes and van der Waals weakened by substrate roughness. Our findings quantify non-specific kT-scale interactions between SLB-modified colloids and surfaces, which enables the design of such systems for use in biomedical applications and studies of biomolecular interactions.

  5. Positively charged supported lipid bilayer formation on gold surfaces for neuronal cell culture.

    PubMed

    Choi, Sung-Eun; Greben, Kyrylo; Wördenweber, Roger; Offenhäusser, Andreas

    2016-06-06

    Supported lipid bilayers are widely used as cell membrane models and sensor platforms, but the usage on gold surface needs additional surface modification or optimized experimental conditions. In this work, the authors show lipid bilayer formation on plasma activated gold surfaces in physiological conditions without any other modification if at least 30% positively charged lipids are present. Details of bilayer formation from small unilamellar vesicles were monitored using quartz crystal microbalance with dissipation in both basic and acidic environment. The authors also confirmed that this positively charged bilayer system can sustain primary cortical neuron growth and lipid transfer. This method will provide simple means to construct biomimetic interface on gold electrodes.

  6. Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

    PubMed Central

    Cukierman, S; Quigley, E P; Crumrine, D S

    1997-01-01

    concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers. PMID:9370442

  7. Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

    PubMed

    Cukierman, S; Quigley, E P; Crumrine, D S

    1997-11-01

    concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.

  8. Interaction of curcumin with lipid monolayers and liposomal bilayers.

    PubMed

    Karewicz, Anna; Bielska, Dorota; Gzyl-Malcher, Barbara; Kepczynski, Mariusz; Lach, Radosław; Nowakowska, Maria

    2011-11-01

    Curcumin shows huge potential as an anticancer and anti-inflammatory agent. However, to achieve a satisfactory bioavailability and stability of this compound, its liposomal form is preferable. Our detailed studies on the curcumin interaction with lipid membranes are aimed to obtain better understanding of the mechanism and eventually to improve the efficiency of curcumin delivery to cells. Egg yolk phosphatidylcholine (EYPC) one-component monolayers and bilayers, as well as mixed systems containing additionally dihexadecyl phosphate (DHP) and cholesterol, were studied. Curcumin binding constant to EYPC liposomes was determined based on two different methods: UV/Vis absorption and fluorescence measurements to be 4.26×10(4)M(-1) and 3.79×10(4)M(-1), respectively. The fluorescence quenching experiment revealed that curcumin locates in the hydrophobic region of EYPC liposomal bilayer. It was shown that curcumin impacts the size and stability of the liposomal carriers significantly. Loaded into the EYPC/DPH/cholesterol liposomal bilayer curcumin stabilizes the system proportionally to its content, while the EYPC/DPH system is destabilized upon drug loading. The three-component lipid composition of the liposome seems to be the most promising system for curcumin delivery. An interaction of free and liposomal curcumin with EYPC and mixed monolayers was also studied using Langmuir balance measurements. Monolayer systems were treated as a simple model of cell membrane. Condensing effect of curcumin on EYPC and EYPC/DHP monolayers and loosening influence on EYPC/DHP/chol ones were observed. It was also demonstrated that curcumin-loaded EYPC liposomes are more stable upon interaction with the model lipid membrane than the unloaded ones.

  9. Assembly of RNA nanostructures on supported lipid bilayers.

    PubMed

    Dabkowska, Aleksandra P; Michanek, Agnes; Jaeger, Luc; Rabe, Michael; Chworos, Arkadiusz; Höök, Fredrik; Nylander, Tommy; Sparr, Emma

    2015-01-14

    The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.

  10. Assembly of RNA nanostructures on supported lipid bilayers

    PubMed Central

    Dabkowska, Aleksandra P.; Michanek, Agnes; Jaeger, Luc; Rabe, Michael; Chworos, Arkadiusz; Höök, Fredrik; Nylander, Tommy; Sparr, Emma

    2014-01-01

    The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nanostructures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces. PMID:25417592

  11. Lipid Bilayer Vesicle Dynamics in AC Electric Fields

    NASA Astrophysics Data System (ADS)

    McConnell, Lane; Vlahovska, Petia; Miksis, Michael

    2014-11-01

    Vesicles are closed, fluid-filled lipid bilayers which are mechanically similar to biological cells and which undergo shape transitions in the presence of electric fields. Here we model the vesicle membrane as an infinitely thin, capacitive, area-incompressible interface with the surrounding fluids acting as charge-advecting leaky dielectrics. We then implement the boundary integral method to numerically investigate the dynamics of a vesicle in various AC electric field profiles. Our numerical results are then compared with recent small deformation theory and experimental data. We also note our observation of a new theoretical vesicle behavior that has yet to be observed experimentally.

  12. Lipid bilayer array for simultaneous recording of ion channel activities

    NASA Astrophysics Data System (ADS)

    Hirano-Iwata, Ayumi; Nasu, Tomohiro; Oshima, Azusa; Kimura, Yasuo; Niwano, Michio

    2012-07-01

    This paper describes an array of stable and reduced-solvent bilayer lipid membranes (BLMs) formed in microfabricated silicon chips. BLMs were first vertically formed simultaneously and then turned 90° in order to realize a horizontal BLM array. Since the present BLMs are mechanically stable and robust, the BLMs survive this relatively tough process. Typically, a ˜60% yield in simultaneous BLM formation over 9 sites was obtained. Parallel recordings of gramicidin channel activities from different BLMs were demonstrated. The present system has great potential as a platform of BLM-based high throughput drug screening for ion channel proteins.

  13. Kinematics, material symmetry, and energy densities for lipid bilayers with spontaneous curvature

    PubMed Central

    Maleki, Mohsen; Seguin, Brian; Fried, Eliot

    2013-01-01

    Continuum mechanical tools are used to describe the deformation, energy density, and material symmetry of a lipid bilayer with spontaneous curvature. In contrast to conventional approaches in which lipid bilayers are modeled by material surfaces, here we rely on a three-dimensional approach in which a lipid bilayer is modeling by a shell-like body with finite thickness. In this setting, the interface between the leaflets of a lipid bilayer is assumed to coincide with the mid-surface of the corresponding shell-like body. The three-dimensional deformation gradient is found to involve the curvature tensors of the mid-surface in the spontaneous and the deformed states, the deformation gradient of the mid-surface, and the transverse deformation. Attention is also given to the coherency of the leaflets and to the area compatibility of closed lipid bilayers (i.e., vesicles). A hyperelastic constitutive theory for lipid bilayers in the liquid phase is developed. In combination, the requirements of frame-indifference and material symmetry yield a representation for the energy density of a lipid bilayer. This representation shows that three scalar invariants suffice to describe the constitutive response of a lipid bilayer exhibiting in-plane fluidity and transverse isotropy. In addition to exploring the geometrical and physical properties of these invariants, fundamental constitutively-associated kinematical quantities are emphasized. On this basis, the effect on the energy density of assuming that the lipid bilayer is incompressible is considered. Lastly, a dimension reduction argument is used to extract an areal energy density per unit area from the three-dimensional energy density. This step explains the origin of spontaneous curvature in the areal energy density. Importantly, along with a standard contribution associated with the natural curvature of lipid bilayer, our analysis indicates that constitutive asymmetry between the leaflets of the lipid bilayer gives rise to

  14. Anisotropic metal growth on phospholipid nanodiscs via lipid bilayer expansion

    NASA Astrophysics Data System (ADS)

    Oertel, Jana; Keller, Adrian; Prinz, Julia; Schreiber, Benjamin; Hübner, René; Kerbusch, Jochen; Bald, Ilko; Fahmy, Karim

    2016-05-01

    Self-assembling biomolecules provide attractive templates for the preparation of metallic nanostructures. However, the intuitive transfer of the “outer shape” of the assembled macromolecules to the final metallic particle depends on the intermolecular forces among the biomolecules which compete with interactions between template molecules and the metal during metallization. The shape of the bio-template may thus be more dynamic than generally assumed. Here, we have studied the metallization of phospholipid nanodiscs which are discoidal particles of ~10 nm diameter containing a lipid bilayer ~5 nm thick. Using negatively charged lipids, electrostatic adsorption of amine-coated Au nanoparticles was achieved and followed by electroless gold deposition. Whereas Au nanoparticle adsorption preserves the shape of the bio-template, metallization proceeds via invasion of Au into the hydrophobic core of the nanodisc. Thereby, the lipidic phase induces a lateral growth that increases the diameter but not the original thickness of the template. Infrared spectroscopy reveals lipid expansion and suggests the existence of internal gaps in the metallized nanodiscs, which is confirmed by surface-enhanced Raman scattering from the encapsulated lipids. Interference of metallic growth with non-covalent interactions can thus become itself a shape-determining factor in the metallization of particularly soft and structurally anisotropic biomaterials.

  15. Anisotropic metal growth on phospholipid nanodiscs via lipid bilayer expansion

    PubMed Central

    Oertel, Jana; Keller, Adrian; Prinz, Julia; Schreiber, Benjamin; Hübner, René; Kerbusch, Jochen; Bald, Ilko; Fahmy, Karim

    2016-01-01

    Self-assembling biomolecules provide attractive templates for the preparation of metallic nanostructures. However, the intuitive transfer of the “outer shape” of the assembled macromolecules to the final metallic particle depends on the intermolecular forces among the biomolecules which compete with interactions between template molecules and the metal during metallization. The shape of the bio-template may thus be more dynamic than generally assumed. Here, we have studied the metallization of phospholipid nanodiscs which are discoidal particles of ~10 nm diameter containing a lipid bilayer ~5 nm thick. Using negatively charged lipids, electrostatic adsorption of amine-coated Au nanoparticles was achieved and followed by electroless gold deposition. Whereas Au nanoparticle adsorption preserves the shape of the bio-template, metallization proceeds via invasion of Au into the hydrophobic core of the nanodisc. Thereby, the lipidic phase induces a lateral growth that increases the diameter but not the original thickness of the template. Infrared spectroscopy reveals lipid expansion and suggests the existence of internal gaps in the metallized nanodiscs, which is confirmed by surface-enhanced Raman scattering from the encapsulated lipids. Interference of metallic growth with non-covalent interactions can thus become itself a shape-determining factor in the metallization of particularly soft and structurally anisotropic biomaterials. PMID:27216789

  16. Anisotropic metal growth on phospholipid nanodiscs via lipid bilayer expansion.

    PubMed

    Oertel, Jana; Keller, Adrian; Prinz, Julia; Schreiber, Benjamin; Hübner, René; Kerbusch, Jochen; Bald, Ilko; Fahmy, Karim

    2016-01-01

    Self-assembling biomolecules provide attractive templates for the preparation of metallic nanostructures. However, the intuitive transfer of the "outer shape" of the assembled macromolecules to the final metallic particle depends on the intermolecular forces among the biomolecules which compete with interactions between template molecules and the metal during metallization. The shape of the bio-template may thus be more dynamic than generally assumed. Here, we have studied the metallization of phospholipid nanodiscs which are discoidal particles of ~10 nm diameter containing a lipid bilayer ~5 nm thick. Using negatively charged lipids, electrostatic adsorption of amine-coated Au nanoparticles was achieved and followed by electroless gold deposition. Whereas Au nanoparticle adsorption preserves the shape of the bio-template, metallization proceeds via invasion of Au into the hydrophobic core of the nanodisc. Thereby, the lipidic phase induces a lateral growth that increases the diameter but not the original thickness of the template. Infrared spectroscopy reveals lipid expansion and suggests the existence of internal gaps in the metallized nanodiscs, which is confirmed by surface-enhanced Raman scattering from the encapsulated lipids. Interference of metallic growth with non-covalent interactions can thus become itself a shape-determining factor in the metallization of particularly soft and structurally anisotropic biomaterials. PMID:27216789

  17. Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers.

    PubMed Central

    Dumas, F; Sperotto, M M; Lebrun, M C; Tocanne, J F; Mouritsen, O G

    1997-01-01

    A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and

  18. Lipid bilayer membrane affinity rationalizes inhibition of lipid peroxidation by a natural lignan antioxidant.

    PubMed

    Podloucká, Pavlína; Berka, Karel; Fabre, Gabin; Paloncýová, Markéta; Duroux, Jean-Luc; Otyepka, Michal; Trouillas, Patrick

    2013-05-01

    Lipid peroxidation is a degenerative oxidative process that modifies the structure of membranes, influencing their biological functions. Lignans, natural polyphenolic antioxidants widely distributed in plants, can prevent this membrane damage by free-radical scavenging. Here, we rationalize the difference in lipid peroxidation inhibition activity of argenteane, a natural dilignan isolated from wild nutmeg, and 3,3'-dimethoxy-1,1'-biphenyl-2,2'-diol, which represents the central part of argenteane responsible for its antioxidant activity. Although both compounds have the same capacity to scavenge free radicals, argenteane is a more active inhibitor of lipid peroxidation. We show that both compounds penetrate into DOPC and PLPC lipid bilayers and adopt similar positions and orientations, which therefore does not explain the difference in their lipid peroxidation inhibition activity. However, free energy profiles indicate that argenteane has a significantly higher affinity to the lipid bilayer, and thus a higher effective concentration to scavenge radicals formed during lipid peroxidation. This finding explains the higher activity of argenteane to inhibit lipid peroxidation. PMID:23560800

  19. Supported lipid bilayer membranes for water purification by reverse osmosis.

    PubMed

    Kaufman, Yair; Berman, Amir; Freger, Viatcheslav

    2010-05-18

    Some biological plasma membranes pass water with a permeability and selectivity largely exceeding those of commercial membranes for water desalination using specialized trans-membrane proteins aquaporins. However, highly selective transport of water through aquaporins is usually driven by an osmotic rather mechanical pressure, which is not as attractive from the engineering point of view. The feasibility of adopting biomimetic membranes for water purification driven by a mechanical pressure, i.e., filtration is explored in this paper. Toward this goal, it is proposed to use a commercial nanofiltration (NF) membrane as a support for biomimetic lipid bilayer membranes to render them robust enough to withstand the required pressures. It is shown in this paper for the first time that by properly tuning molecular interactions supported phospholipid bilayers (SPB) can be prepared on a commercial NF membrane. The presence of SPB on the surface was verified and quantified by several spectroscopic and microscopic techniques, which showed morphology close to the desired one with very few defects. As an ultimate test it is shown that hydraulic permeability of the SPB supported on the NF membrane (NTR-7450) approaches the values deduced from the typical osmotic permeabilities of intact continuous bilayers. This permeability was unaffected by the trans-membrane flow of water and by repeatedly releasing and reapplying a 10 bar pressure. Along with a parallel demonstration that aquaporins could be incorporated in a similar bilayer on mica, this demonstrates the feasibility of the proposed approach. The prepared SPB structure may be used as a platform for preparing biomimetic filtration membranes with superior performance based on aquaporins. The concept of SPBs on permeable substrates of the present type may also be useful in the future for studying transport of various molecules through trans-membrane proteins. PMID:20099798

  20. Supported lipid bilayer membranes for water purification by reverse osmosis.

    PubMed

    Kaufman, Yair; Berman, Amir; Freger, Viatcheslav

    2010-05-18

    Some biological plasma membranes pass water with a permeability and selectivity largely exceeding those of commercial membranes for water desalination using specialized trans-membrane proteins aquaporins. However, highly selective transport of water through aquaporins is usually driven by an osmotic rather mechanical pressure, which is not as attractive from the engineering point of view. The feasibility of adopting biomimetic membranes for water purification driven by a mechanical pressure, i.e., filtration is explored in this paper. Toward this goal, it is proposed to use a commercial nanofiltration (NF) membrane as a support for biomimetic lipid bilayer membranes to render them robust enough to withstand the required pressures. It is shown in this paper for the first time that by properly tuning molecular interactions supported phospholipid bilayers (SPB) can be prepared on a commercial NF membrane. The presence of SPB on the surface was verified and quantified by several spectroscopic and microscopic techniques, which showed morphology close to the desired one with very few defects. As an ultimate test it is shown that hydraulic permeability of the SPB supported on the NF membrane (NTR-7450) approaches the values deduced from the typical osmotic permeabilities of intact continuous bilayers. This permeability was unaffected by the trans-membrane flow of water and by repeatedly releasing and reapplying a 10 bar pressure. Along with a parallel demonstration that aquaporins could be incorporated in a similar bilayer on mica, this demonstrates the feasibility of the proposed approach. The prepared SPB structure may be used as a platform for preparing biomimetic filtration membranes with superior performance based on aquaporins. The concept of SPBs on permeable substrates of the present type may also be useful in the future for studying transport of various molecules through trans-membrane proteins.

  1. Unraveling lipid/protein interaction in model lipid bilayers by Atomic Force Microscopy.

    PubMed

    Alessandrini, Andrea; Facci, Paolo

    2011-01-01

    The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane-associated proteins, favoring protein-protein interaction and influence signaling and trafficking. The Atomic Force Microscope allows to study the localization of membrane-associated proteins with respect to the lipid organization at the single molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating in particular on GPI-anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will also discuss the functional consequences of what has been imaged by Atomic Force Microscopy.

  2. The pathological prion protein forms ionic conductance in lipid bilayer.

    PubMed

    Paulis, Daniele; Maras, Bruno; Schininà, M Eugenia; di Francesco, Laura; Principe, Serena; Galeno, Roberta; Abdel-Haq, Hanin; Cardone, Franco; Florio, Tullio; Pocchiari, Maurizio; Mazzanti, Michele

    2011-08-01

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP(TSE)). PrP(TSE) pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP(TSE) on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP(TSE) isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.

  3. Assembly of RNA nanostructures on supported lipid bilayers

    NASA Astrophysics Data System (ADS)

    Dabkowska, Aleksandra P.; Michanek, Agnes; Jaeger, Luc; Rabe, Michael; Chworos, Arkadiusz; Höök, Fredrik; Nylander, Tommy; Sparr, Emma

    2014-12-01

    The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of

  4. Theoretical studies of lipid bilayer electroporation using molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Levine, Zachary Alan

    Computer simulations of physical, chemical, and biological systems have improved tremendously over the past five decades. From simple studies of liquid argon in the 1960s to fully atomistic simulations of entire viruses in the past few years, recent advances in high-performance computing have continuously enabled simulations to bridge the gap between scientific theory and experiment. Molecular dynamics simulations in particular have allowed for the direct observation of spatial and temporal events which are at present inaccessible to experiments. For this dissertation I employ all-atom molecular dynamics simulations to study the transient, electric field-induced poration (or electroporation) of phospholipid bilayers at MV/m electric fields. Phospholipid bilayers are the dominant constituents of cell membranes and act as both a barrier and gatekeeper to the cell interior. This makes their structural integrity and susceptibility to external perturbations an important topic for study, especially as the density of electromagnetic radiation in our environment is increasing steadily. The primary goal of this dissertation is to understand the specific physical and biological mechanisms which facilitate electroporation, and to connect our simulated observations to experiments with live cells and to continuum models which seek to describe the underlying biological processes of electroporation. In Chapter 1 I begin with a brief introduction to phospholipids and phospholipid bilayers, followed by an extensive overview of electroporation and atomistic molecular dynamics simulations. The following chapters will then focus on peer-reviewed and published work we performed, or on existing projects which are currently being prepared for submission. Chapter 2 looks at how external electric fields affect both oxidized and unoxidized lipid bilayers as a function of oxidation concentration and oxidized lipid type. Oxidative damage to cell membranes represents a physiologically relevant

  5. Investigating Hydrophilic Pores in Model Lipid Bilayers Using Molecular Simulations: Correlating Bilayer Properties with Pore-Formation Thermodynamics.

    PubMed

    Hu, Yuan; Sinha, Sudipta Kumar; Patel, Sandeep

    2015-06-23

    Cell-penetrating and antimicrobial peptides show a remarkable ability to translocate across physiological membranes. Along with factors such as electric-potential-induced perturbations of membrane structure and surface tension effects, experiments invoke porelike membrane configurations during the solute transfer process into vesicles and cells. The initiation and formation of pores are associated with a nontrivial free-energy cost, thus necessitating a consideration of the factors associated with pore formation and the attendant free energies. Because of experimental and modeling challenges related to the long time scales of the translocation process, we use umbrella sampling molecular dynamics simulations with a lipid-density-based order parameter to investigate membrane-pore-formation free energy employing Martini coarse-grained models. We investigate structure and thermodynamic features of the pore in 18 lipids spanning a range of headgroups, charge states, acyl chain lengths, and saturation. We probe the dependence of pore-formation barriers on the area per lipid, lipid bilayer thickness, and membrane bending rigidities in three different lipid classes. The pore-formation free energy in pure bilayers and peptide translocating scenarios are significantly coupled with bilayer thickness. Thicker bilayers require more reversible work to create pores. The pore-formation free energy is higher in peptide-lipid systems than in peptide-free lipid systems due to penalties to maintain the solvation of charged hydrophilic solutes within the membrane environment.

  6. Permeability of lipid bilayers to amino acids and phosphate

    NASA Technical Reports Server (NTRS)

    Chakrabarti, A. C.; Deamer, D. W.

    1992-01-01

    Permeability coefficients for amino acid classes, including neutral, polar, hydrophobic, and charged species, were measured and compared with values for other ionic solutes such as phosphate. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (EPC) or dimyristoylphosphatidylcholine (DMPC). The following permeability coefficients were obtained for: glycine, 5.7 x 10(-12) cm s-1 (EPC), 2.0 x 10(-11) cm s-1 (DMPC); serine, 5.5 x 10(-12) cm s-1 (EPC), 1.6 x 10(-11) cm s-1 (DMPC); lysine, 5.1 x 10(-12) cm s-1 (EPC), 1.9 x 10(-11) cm s-1 (DMPC); tryptophan, 4.1 x 10(-10) cm s-1 (EPC); and phenylalanine, 2.5 x 10(-10) cm s-1 (EPC). Decreasing lipid chain length increased permeability slightly, while variations in pH had only minor effects on the permeability coefficients of the amino acids tested. Phosphate permeability was in the range of 10(-12)-10(-13) cm s-1 depending on the pH of the medium. The values for the polar and charged amino acids were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium, which are in the range of 10(-12)-10(-13) cm s-1, depending on conditions and the lipid species used. This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. The results are relevant to the permeation of certain peptides into lipid bilayers during protein translocation and membrane biogenesis.

  7. Tethered or adsorbed supported lipid bilayers in nanotubes characterized by deuterium magic angle spinning NMR spectroscopy.

    PubMed

    Wattraint, Olivier; Warschawski, Dror E; Sarazin, Catherine

    2005-04-12

    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. PMID:15807556

  8. Corrugation of Phase-Separated Lipid Bilayers Supported by Nanoporous Silica Xerogel Surfaces

    SciTech Connect

    Goksu, E I; Nellis, B A; Lin, W; Satcher Jr., J H; Groves, J T; Risbud, S H; Longo, M L

    2008-10-30

    Lipid bilayers supported by substrates with nanometer-scale surface corrugations holds interest in understanding both nanoparticle-membrane interactions and the challenges of constructing models of cell membranes on surfaces with desirable properties, e.g. porosity. Here, we successfully form a two-phase (gel-fluid) lipid bilayer supported by nanoporous silica xerogel. Surface topology, diffusion, and lipid density in comparison to mica-supported lipid bilayers were characterized by AFM, FRAP, FCS, and quantitative fluorescence microscopy, respectively. We found that the two-phase lipid bilayer follows the xerogel surface contours. The corrugation imparted on the lipid bilayer results in a lipid density that is twice that on a flat mica surface. In direct agreement with the doubling of actual bilayer area in a projected area, we find that the lateral diffusion coefficient (D) of lipids on xerogel ({approx}1.7 {micro}m{sup 2}/s) is predictably lower than on mica ({approx}4.1 {micro}m{sup 2}/s) by both FRAP and FCS techniques. Furthermore, the gel-phase domains on xerogel compared to mica were larger and less numerous. Overall, our results suggest the presence of a relatively defect-free continuous two-phase bilayer that penetrates approximately midway into the first layer of {approx}50 nm xerogel beads.

  9. Phase separation in lipid bilayers triggered by low pH

    SciTech Connect

    Suresh, Swetha; Edwardson, J. Michael

    2010-09-03

    Research highlights: {yields} Lipid bilayers have been imaged by atomic force microscopy (AFM). {yields} At pH 5 phase separation occurs in lipid bilayers containing mixed acyl chains. {yields} Phase separation does not occur when lipids have only unsaturated chains. {yields} Phase separation might drive protein clustering during endocytosis. -- Abstract: Endocytosis involves the capture of membrane from the cell surface in the form of vesicles, which become rapidly acidified to about pH 5. Here we show using atomic force microscopy (AFM) imaging that this degree of acidification triggers phase separation in lipid bilayers containing mixed acyl chains (e.g. palmitoyl/oleoyl) or complex mixtures (e.g. total brain extract) but not in bilayers containing only lipids with unsaturated chains (e.g. dioleoyl). Since mixed-chain lipids are major constituents of the outer leaflet of the plasma membrane, the type of phase separation reported here might support protein clustering and signaling during endocytosis.

  10. Lipid reassembly in asymmetric Langmuir-Blodgett/Langmuir-Schaeffer bilayers.

    PubMed

    Yuan, Jie; Hao, Changchun; Chen, Maohui; Berini, Pierre; Zou, Shan

    2013-01-01

    Molecular-reorganization-induced morphology alteration in asymmetric substrate-supported lipid bilayers (SLBs) was directly visualized by means of atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) microscopy. SLB samples were fabricated on mica-on-glass and glass substrates by Langmuir-Blodgett (LB)/Langmuir-Schaeffer (LS) using binary lipid mixtures, namely, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and ternary mixtures DOPC/DPPC/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), labeled with 0.2 mol % Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (TR-DHPE) dye. Phase segregations were characterized by TIRF imaging, and DPPC-enriched domain structures were also observed. Interestingly for ∼40% (n = 6) of the samples with binary mixtures in the LB leaflet and a single component in the LS leaflet, that is, (DOPC/DPPC)(LB)+DOPC(LS), the contrast of the DPPC domains changed from the original dark (without dye) to bright (more TR dye partitioning) on TIRF images, returning to dark again. This contrast reverse was also correlated to AFM height images, where a DPPC-DPPC gel phase was spotted after the TIRF image contrast returned to dark. The rupture force mapping results measured on these binary mixture samples also confirmed unambiguously the formation of DPPC-DPPC gel domain components during the contrast change. The samples were tracked over 48 h to investigate the lipid molecule movements in both the DPPC domains and the DOPC fluid phase. The fluorescence contrast changes from bright to dark in SLBs indicate that the movement of dye molecules was independent of the movement of lipid molecules. In addition, correlated multimodal imaging using AFM, force mapping, and fluorescence provides a novel route to uncover the reorganization of lipid molecules at the solid-liquid interface, suggesting that the dynamics of dye molecules is highly

  11. Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film.

    PubMed

    Choi, Sangbaek; Yoon, Sunhee; Ryu, Hyunil; Kim, Sun Min; Jeon, Tae-Joon

    2016-01-01

    An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 °C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of < 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system. PMID:27501114

  12. Permeability of small nonelectrolytes through lipid bilayer membranes

    SciTech Connect

    Walter, A.; Gutknecht, J.

    1986-01-01

    Diffusion of small nonelectrolytes through planar lipid bilayer membranes (egg phosphatidylcholine-decane) was examined by correlating the permeability coefficients of 22 solutes with their partition coefficients between water and four organic solvents. High correlations were observed with hexadecane and olive oil (r = 0.95 and 0.93), but not octanol and ether (r = 0.75 and 0.74). Permeabilities of the seven smallest molecules (mol wt less than 50) (water, hydrofluoric acid, hydrochloric acid, ammonia, methylamine, formic acid and formamide) were 2- to 15-fold higher than the values predicted by the permeabilities of the larger molecules (50 less than mol wt less than 300). The extra permeabilities of the seven smallest molecules were not correlated with partition coefficients but were inversely correlated with molecular volumes. The larger solute permeabilities also decreased with increasing molecular volume, but the relationship was neither steep nor significant. The permeability pattern cannot be explained by the molecular volume dependence of partitioning into the bilayer or by the existence of transient aqueous pores. The molecular volume dependence of solute permeability suggests that the membrane barrier behaves more like a polymer than a liquid hydrocarbon. All the data are consistent with the solubility-diffusion model, which can explain both the hydrophobicity dependence and the molecular volume dependence of nonelectrolyte permeability.

  13. Lipid monolayer states and their relationships to bilayers.

    PubMed Central

    MacDonald, R C; Simon, S A

    1987-01-01

    Uncommon methods of formation and analysis of lipid monolayers have enabled the recognition of several monolayer states and the identification of that in which molecular organization corresponds closely to that of the bilayer. Monolayers were formed by continuously adding a solution of phospholipid [dimyristoyl phosphatidylcholine in hexane/ethanol, 9:1 (vol/vol)] to the air/water interface of a constant-area trough. This procedure generates unconventional surface pressure (pi)-surface concentration (gamma) isotherms, which for liquid-crystalline monolayers consist of straight lines with three prominent intersections, two of which are not apparent in conventional pi-A isotherms. The regions of linear change of pi are explicable in terms of the area dependence of alkyl chain entropy. The two breaks at lower pi delimit states in which both chains lie parallel to the surface. The third occurs at collapse, which corresponds to a true equilibrium for unstressed liposomes. Mechanical and thermodynamic properties of bilayers, particularly phase-transition parameters, correspond closely to those of monolayers with which they are in equilibrium. Images PMID:3473494

  14. Kinetics of Domains Registration in Multicomponent Lipid Bilayer Membranes

    PubMed Central

    Sornbundit, Kan; Modchang, Charin; Triampo, Wannapong; Triampo, Darapond; Nuttavut, Narin; Sunil Kumar, P.B; Laradji, Mohamed

    2014-01-01

    The kinetics of registration of lipid domains in the apposing leaflets of symmetric bilayer membranes is investigated via systematic dissipative particle dynamics simulations. The decay of the distance between the centres of mass of the domains in the apposing leaflets is almost linear during early stages, and then becomes exponential during late times. The time scales of both linear and exponential decays are found to increase with decreasing the strength of interleaflet coupling. The ratio between the time scales of the exponential and linear regimes decreases with increasing the domain size, implying that the decay of the distance between the domains centres of mass is essentially linear for large domains. These numerical results are largely in agreement with the recent theoretical predictions of Han and Haataja [Soft Matter (2013) 9:2120-2124]. We also found that the domains become elongated during the registration process. PMID:25090030

  15. Hydrofluoric and nitric acid transport through lipid bilayer membranes.

    PubMed

    Gutknecht, J; Walter, A

    1981-06-01

    Hydrofluoric and nitric acid transport through lipid bilayer membranes were studied by a combination of electrical conductance and pH electrode techniques. Transport occurs primarily by nonionic diffusion of molecular HF and HNO3. Membrane permeabilities to HF and HNO3 ranged from 10(-4) to 10(-3) cm . s-1, five to seven orders of magnitude higher than the permeabilities to NO-3, F- and H+. Our results are consistent with the hypothesis that F- transport through biological membranes occurs mainly by nonionic diffusion of HF. Our results also suggest that of the two principal components of 'acid rain', HNO3 may be more toxic than H2SO4.

  16. Simulated microgravity impacts the plant plasmalemma lipid bilayer

    NASA Astrophysics Data System (ADS)

    Nedukha, Olena; Berkovich, Yuliy A.; Vorobyeva, Tamara; Grakhov, Volodimir; Klimenko, Elena; Zhupanov, Ivan; Jadko, Sergiy

    Biological membranes, especially the plasmalemma, and their properties and functions can be considered one of the most sensitive indicators of gravity interaction or alteration of gravity, respectively. Studies on the molecular basis of cellular signal perception and transduction are very important in order to understand signal responses at the cellular and organism level. The plasmalemma lipid bilayer is the boundary between the cell internal and external environment and mediates communication between them. Therefore, we studied the content and composition of lipids, saturated and unsaturated fatty acids, sterols, and microviscosity in the plasmalemma isolated from pea seedling roots and epicotyls grown in the stationary conditions and under slow horizontal clinorotation. In addition, lipid peroxidation intensity of intact roots was also identified. The plasmalemma fraction was isolated by the two-phase aquatic-polymer system optimized for pea using a centrifuge Optima L-90K. Lipid bilayer components were determined by using highly effective liquid chromatography with a system Angilent 1100 (Germany). Spontaneous chemiluminescence intensity was measured with a chemiluminometer ChLMTS-01. The obtained data showed that plasmalemma investigated parameters are sensitive to clinorotation, namely: increasing or decreasing the different lipids content, among which, phospho- and glycolipids were dominated, as well as changes in the content of saturated and unsaturated fatty acids and sterols. A degree of plasmalemma sensitivity to clinorotation was higher for the root plasmalemma than epicocotyl ones. This distinguish may be naturally explained by the differences in the structure, cell types, growth, and specific functions of a root and an epicotyl, those are the most complicated in roots. An index of unsaturation under clinorotation was similar to that in the stationary conditions as a result of the certain balance between changes in the content of saturated and

  17. New optical method for measuring the bending elasticity of lipid bilayers

    NASA Astrophysics Data System (ADS)

    Minetti, C.; Vitkova, V.; Dubois, F.; Bivas, I.

    2016-02-01

    The knowledge of the elasticity of lipid bilayer structures is fundamental for new developments in biophysics, pharmacology and biomedical research. Lipid vesicles are readily prepared in laboratory conditions and employed for studying the physical properties of lipid membranes. The thermal fluctuation analysis of the shape of lipid vesicles (or flicker spectroscopy) is one of the experimental methods widely used for the measurement of the bending modulus of lipid bilayers. We present direct phase measurements performed on dilute vesicular suspensions by means of a new optical method exploiting holographic microscopy. For the bending constant of phosphatidylcholine bilayers we report the value of 23kBT in agreement with values previously measured by micropipette aspiration, electrodeformation and flicker spectroscopy of giant lipid vesicles. The application of this novel approach for the evaluation of the bending elasticity of lipid membranes opens the way to future developments in the phase measurements on lipid vesicles for the evaluation of their mechanical constants.

  18. High Resistivity Lipid Bilayers Assembled on Polyelectrolyte Multilayer Cushions: An Impedance Study.

    PubMed

    Diamanti, Eleftheria; Gregurec, Danijela; Rodríguez-Presa, María José; Gervasi, Claudio A; Azzaroni, Omar; Moya, Sergio E

    2016-06-28

    Supported membranes on top of polymer cushions are interesting models of biomembranes as cell membranes are supported on a polymer network of proteins and sugars. In this work lipid vesicles formed by a mixture of 30% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 70% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) are assembled on top of a polyelectrolyte multilayer (PEM) cushion of poly(allylamine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS). The assembly results in the formation of a bilayer on top of the PEM as proven by means of the quartz crystal microbalance with dissipation technique (QCM-D) and by cryo-transmission electron microscopy (cryo-TEM). The electrical properties of the bilayer are studied by electrochemical impedance spectroscopy (EIS). The bilayer supported on the PEMs shows a high resistance, on the order of 10(7) Ω cm(2), which is indicative of a continuous, dense bilayer. Such resistance is comparable with the resistance of black lipid membranes. This is the first time that such values are obtained for lipid bilayers supported on PEMs. The assembly of polyelectrolytes on top of a lipid bilayer decreases the resistance of the bilayer up to 2 orders of magnitude. The assembly of the polyelectrolytes on the lipids induces defects or pores in the bilayer which in turn prompts a decrease in the measured resistance.

  19. High Resistivity Lipid Bilayers Assembled on Polyelectrolyte Multilayer Cushions: An Impedance Study.

    PubMed

    Diamanti, Eleftheria; Gregurec, Danijela; Rodríguez-Presa, María José; Gervasi, Claudio A; Azzaroni, Omar; Moya, Sergio E

    2016-06-28

    Supported membranes on top of polymer cushions are interesting models of biomembranes as cell membranes are supported on a polymer network of proteins and sugars. In this work lipid vesicles formed by a mixture of 30% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 70% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) are assembled on top of a polyelectrolyte multilayer (PEM) cushion of poly(allylamine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS). The assembly results in the formation of a bilayer on top of the PEM as proven by means of the quartz crystal microbalance with dissipation technique (QCM-D) and by cryo-transmission electron microscopy (cryo-TEM). The electrical properties of the bilayer are studied by electrochemical impedance spectroscopy (EIS). The bilayer supported on the PEMs shows a high resistance, on the order of 10(7) Ω cm(2), which is indicative of a continuous, dense bilayer. Such resistance is comparable with the resistance of black lipid membranes. This is the first time that such values are obtained for lipid bilayers supported on PEMs. The assembly of polyelectrolytes on top of a lipid bilayer decreases the resistance of the bilayer up to 2 orders of magnitude. The assembly of the polyelectrolytes on the lipids induces defects or pores in the bilayer which in turn prompts a decrease in the measured resistance. PMID:27267089

  20. Dynamics, Surface Electrostatics and Phase Properties of Nanoscale Curved Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Koolivand, Amir

    Surface electrostatic potential of a lipid bilayer governs many vital functions of living cells. Several classes of proteins are known of exhibiting strong binding preferences to curved lipid bilayer surfaces. In this project we employed electron paramagnetic resonance (EPR) of a recently introduced phospholipid (IMTSL-PTE) bearing a pH-sensitive nitroxide covalently attached to the lipid head group to measure the surface electrostatics of the lipid membrane and nanopore-confined lipid bilayers as a function of the bilayer curvature. The pKa of the ionizable group of this lipid-based spin probe is reporting on the bilayer surface electrostatics potential by changes in the EPR spectra. Specifically, both rotational dynamics and magnetic parameters of the nitroxide are affected by the probe protonation. Effect of curvature on the surface electrostatic potential and dynamics of lipid bilayer was studied for POPG and DMPG unilamellar vesicles (ULVs). It was found that the magnitude of the negative surface electrostatic potential increased upon decrease in the vesicle diameter for the bilayers in the fluid phase; however, no significant changes were observed for DMPG ULVs in a gel phase. We speculate that biologically relevant fluid bilayer phase allows for a larger variability in the lipid packing density in the lipid polar head group region than a more ordered gel phase and it is likely that the lipid flip-flop is responsible for pH equilibration of IMTSL-PTE. The kinetic EPR study of nitroxide reduction showed that the rate of flip-flop is in the order of 10-5 s-1. The flip-flop rate constant increases when vesicle size deceases. Oxygen permeability measured by X-ban EPR decreases in higher curved vesicles---an observation that is consistent with a tighter packing in smaller vesicles. Partitioning of a small nitroxide molecule TEMPO into ULVs was measured by X-band (9 GHz) and W-band (95 GHz) EPR spectroscopy. The partitioning coefficient of this probe in the lipid

  1. Topologically-Mediated Membrane Dynamics in Supported Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Gilmore, Sean Fitzpatrick

    2011-12-01

    This thesis is primarily design driven. It describes the development and application of dynamically tunable class of solid-fluid interfaces, which serves as a test-bed configuration for fundamental studies of soft condensed matter in reduced dimension. My specific focus is in developing these interfaces to recapitulate topology-mediated phenomenon in biological lipid membranes. The phenomena that the interfacial topology manifest in diffusional characteristics in model membranes are probed using wide-area epifluorescence microscopy and a semi-quantitative analysis of dynamic recovery following photobleaching. Furthermore, real-time remodeling of the membrane-substrate interface topology is shown to provide fundamental information regarding curvature-dependent molecular sorting and resorting. Specifically our experiments using putative raft composition mixtures confirm the conformation-dependent alignment of liquid-ordered domains and moreover reveal domain-domain interactions for the first time in model bilayers. Ongoing work aimed at delineating these inter-domain interactions in terms of membrane elastic properties is being performed. Future work that includes peptide-driven membrane deformation and sorting, as well large-scale, curvature-driven in vivo sorting of lipids is proposed and discussed.

  2. Oligomeric Structure of Colicin Ia Channel in Lipid Bilayer Membranes*

    PubMed Central

    Greig, Sarah L.; Radjainia, Mazdak; Mitra, Alok K.

    2009-01-01

    Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a ∼17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a ∼35 Å-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening. PMID:19357078

  3. Shape control of lipid bilayer membranes by confined actin bundles.

    PubMed

    Tsai, Feng-Ching; Koenderink, Gijsje Hendrika

    2015-12-01

    In living cells, lipid membranes and biopolymers determine each other's conformation in a delicate force balance. Cellular polymers such as actin filaments are strongly confined by the plasma membrane in cell protrusions such as lamellipodia and filopodia. Conversely, protrusion formation is facilitated by actin-driven membrane deformation and these protrusions are maintained by dense actin networks or bundles of actin filaments. Here we investigate the mechanical interplay between actin bundles and lipid bilayer membranes by reconstituting a minimal model system based on cell-sized liposomes with encapsulated actin filaments bundled by fascin. To address the competition between the deformability of the membrane and the enclosed actin bundles, we tune the bundle stiffness (through the fascin-to-actin molar ratio) and the membrane rigidity (through protein decoration). Using confocal microscopy and quantitative image analysis, we show that actin bundles deform the liposomes into a rich set of morphologies. For liposomes having a small membrane bending rigidity, the actin bundles tend to generate finger-like membrane protrusions that resemble cellular filopodia. Stiffer bundles formed at high crosslink density stay straight in the liposome body, whereas softer bundles formed at low crosslink density are bent and kinked. When the membrane has a large bending rigidity, membrane protrusions are suppressed. In this case, membrane enclosure forces the actin bundles to organize into cortical rings, to minimize the energy cost associated with filament bending. Our results highlight the importance of taking into account mechanical interactions between the actin cytoskeleton and the membrane to understand cell shape control.

  4. Analytical investigation of bilayer lipid biosensor based on graphene.

    PubMed

    Akbari, Elnaz; Buntat, Zolkafle; Shahraki, Elmira; Parvaz, Ramtin; Kiani, Mohammad Javad

    2016-01-01

    Graphene is another allotrope of carbon with two-dimensional monolayer honeycomb. Owing to its special characteristics including electrical, physical and optical properties, graphene is known as a more suitable candidate compared to other materials to be used in the sensor application. It is possible, moreover, to use biosensor by using electrolyte-gated field effect transistor based on graphene (GFET) to identify the alterations in charged lipid membrane properties. The current article aims to show how thickness and charges of a membrane electric can result in a monolayer graphene-based GFET while the emphasis is on the conductance variation. It is proposed that the thickness and electric charge of the lipid bilayer (LLP and QLP) are functions of carrier density, and to find the equation relating these suitable control parameters are introduced. Artificial neural network algorithm as well as support vector regression has also been incorporated to obtain other models for conductance characteristic. The results comparison between analytical models, artificial neural network and support vector regression with the experimental data extracted from previous work show an acceptable agreement.

  5. Pore spanning lipid bilayers on silanised nanoporous alumina membranes

    NASA Astrophysics Data System (ADS)

    Md Jani, Abdul M.; Zhou, Jinwen; Nussio, Matthew R.; Losic, Dusan; Shapter, Joe G.; Voelcker, Nicolas H.

    2008-12-01

    The preparation of bilayer lipid membranes (BLMs) on solid surfaces is important for many studies probing various important biological phenomena including the cell barrier properties, ion-channels, biosensing, drug discovery and protein/ligand interactions. In this work we present new membrane platforms based on suspended BLMs on nanoporous anodic aluminium oxide (AAO) membranes. AAO membranes were prepared by electrochemical anodisation of aluminium foil in 0.3 M oxalic acid using a custom-built etching cell and applying voltage of 40 V, at 1oC. AAO membranes with controlled diameter of pores from 30 - 40 nm (top of membrane) and 60 -70 nm (bottom of membrane) were fabricated. Pore dimensions have been confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). AAO membranes were chemically functionalised with 3-aminopropyltriethoxysilane (APTES). Confirmation of the APTES attachment to the AAO membrane was achieved by means of infrared spectroscopy, X-ray photoelectron spectroscopy and contact angle measurements. The Fourier transform infrared (FTIR) spectra of functionalised membranes show several peaks from 2800 to 3000 cm-1 which were assigned to symmetric and antisymmetric CH2 bands. XPS data of the membrane showed a distinct increase in C1s (285 eV), N1s (402 eV) and Si2p (102 eV) peaks after silanisation. The water contact angle of the functionalised membrane was 80o as compared to 20o for the untreated membrane. The formation of BLMs comprising dioleoyl-phosphatidylserine (DOPS) on APTESmodified AAO membranes was carried using the vesicle spreading technique. AFM imaging and force spectroscopy was used to characterise the structural and nanomechanical properties of the suspended membrane. This technique also confirmed the stability of bilayers on the nanoporous alumina support for several days. Fabricated suspended BLMs on nanoporous AAO hold promise for the construction of biomimetic membrane architectures with embedded

  6. Effects of Lipid Composition on Bilayer Membranes Quantified by All-Atom Molecular Dynamics.

    PubMed

    Ding, Wei; Palaiokostas, Michail; Wang, Wen; Orsi, Mario

    2015-12-10

    Biological bilayer membranes typically contain varying amounts of lamellar and nonlamellar lipids. Lamellar lipids, such as dioleoylphosphatidylcholine (DOPC), are defined by their tendency to form the lamellar phase, ubiquitous in biology. Nonlamellar lipids, such as dioleoylphosphatidylethanolamine (DOPE), prefer instead to form nonlamellar phases, which are mostly nonbiological. However, nonlamellar lipids mix with lamellar lipids in biomembrane structures that remain overall lamellar. Importantly, changes in the lamellar vs nonlamellar lipid composition are believed to affect membrane function and modulate membrane proteins. In this work, we employ atomistic molecular dynamics simulations to quantify how a range of bilayer properties are altered by variations in the lamellar vs nonlamellar lipid composition. Specifically, we simulate five DOPC/DOPE bilayers at mixing ratios of 1/0, 3/1, 1/1, 1/3, and 0/1. We examine properties including lipid area and bilayer thickness, as well as the transmembrane profiles of electron density, lateral pressure, electric field, and dipole potential. While the bilayer structure is only marginally altered by lipid composition changes, dramatic effects are observed for the lateral pressure, electric field, and dipole potential profiles. Possible implications for membrane function are discussed.

  7. Formation, Stability, and Mobility of One-Dimensional Lipid Bilayer on High Curvature Substrates

    SciTech Connect

    Huang, J; Martinez, J; Artyukhin, A; Sirbuly, D; Wang, Y; Ju, J W; Stroeve, P; Noy, A

    2007-03-23

    Curved lipid membranes are ubiquitous in living systems and play an important role in many biological processes. To understand how curvature and lipid composition affect membrane formation and fluidity we have assembled and studied mixed 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) supported lipid bilayers on amorphous silicon nanowires with controlled diameters ranging from 20 nm to 200 nm. Addition of cone-shaped DOPE molecules to cylindrical DOPC molecules promotes vesicle fusion and bilayer formation on smaller diameter nanowires. Our experiments demonstrate that nanowire-supported bilayers are mobile, exhibit fast recovery after photobleaching, and have low concentration of defects. Lipid diffusion coefficients in these high-curvature tubular membranes are comparable to the values reported for flat supported bilayers and increase with decreasing nanowire diameter.

  8. Small angle x-ray scattering studies of magnetically oriented lipid bilayers.

    PubMed Central

    Hare, B J; Prestegard, J H; Engelman, D M

    1995-01-01

    Magnetically oriented lipid/detergent bilayers are potentially useful for studies of membrane-associated molecules and complexes using x-ray scattering and nuclear magnetic resonance (NMR). To establish whether the system is a reasonable model of a phospholipid bilayer, we have studied the system using x-ray solution scattering to determine the bilayer thickness, interparticle spacing, and orientational parameters for magnetically oriented lipid bilayers. The magnetically orientable samples contain the phospholipid L-alpha-dilauroylphosphatidylcholine (DLPC) and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) in a 3:1 molar ratio in 70% water (w/v) and are similar to magnetically orientable samples used as NMR media for structural studies of membrane-associated molecules. A bilayer thickness of 30 A was determined for the DLPC/CHAPSO particles, which is the same as the bilayer thickness of pure DLPC vesicles, suggesting that the CHAPSO is not greatly perturbing the lipid bilayer. These data, as well as NMR data on molecules incorporated in the oriented lipid particles, are consistent with the sample consisting of reasonably homogeneous and well dispersed lipid particles. Finally, the orientational energy of the sample suggests that the size of the cooperatively orienting unit in the samples is 2 x 10(7) phospholipid molecules. Images FIGURE 1 PMID:8580332

  9. Hydrodynamic Forces on Macromolecules Protruding from Lipid Bilayers Due to External Liquid Flows.

    PubMed

    Jönsson, Peter; Jönsson, Bengt

    2015-11-24

    It has previously been observed that an externally applied hydrodynamic shear flow above a fluid lipid bilayer can change the local concentration of macromolecules that are associated with the lipid bilayer. The external liquid flow results in a hydrodynamic force on molecules protruding from the lipid bilayer, causing them to move in the direction of the flow. However, there has been no quantitative study about the magnitude of these forces. We here use finite element simulations to investigate how the magnitude of the external hydrodynamic forces varies with the size and shape of the studied macromolecule. The simulations show that the hydrodynamic force is proportional to the effective hydrodynamic area of the studied molecule, Ahydro, multiplied by the mean hydrodynamic shear stress acting on the membrane surface, σhydro. The parameter Ahydro depends on the size and shape of the studied macromolecule above the lipid bilayer and scales with the cross-sectional area of the molecule. We also investigate how hydrodynamic shielding from other surrounding macromolecules decreases Ahydro when the surface coverage of the shielding macromolecules increases. Experiments where the protein streptavidin is anchored to a supported lipid bilayer on the floor of a microfluidic channel were finally performed at three different surface concentrations, Φ = 1%, 6%, and 10%, where the protein is being moved relative to the lipid bilayer by a liquid flow through the channel. From photobleaching measurements of fluorescently labeled streptavidin we found the experimental drift data to be within good accuracy of the simulated results, less than 12% difference, indicating the validity of the results obtained from the simulations. In addition to giving a deeper insight into how a liquid flow can affect membrane-associated molecules in a lipid bilayer, we also see an interesting potential of using hydrodynamic flow experiments together with the obtained results to study the size and

  10. Chronopotentiometric technique as a method for electrical characterization of bilayer lipid membranes.

    PubMed

    Naumowicz, Monika; Figaszewski, Zbigniew Artur

    2011-03-01

    The basic electrical parameters of bilayer lipid membranes are capacitance and resistance. This article describes the application of chronopotentiometry to the research of lipid bilayers. Membranes were made from egg yolk phosphatidylcholine. The chronopotentiometric characteristic of the membranes depends on the current value. For low current values, no electroporation takes place and the voltage rises exponentially to a constant value. Based on these kinds of chronopotentiometric curves, a method of the membrane capacitance and the membrane resistance calculations are presented.

  11. A phenomenological model of the solvent-assisted lipid bilayer formation method.

    PubMed

    Gillissen, Jurriaan J J; Tabaei, Seyed R; Cho, Nam-Joon

    2016-09-21

    The recently introduced solvent-assisted lipid bilayer (SALB) formation method allows one to efficiently fabricate planar, lipid bilayers on solid supports and can be used for various applications. It involves the introduction of an aqueous buffer into a mixture of lipid and alcohol, which is incubated on a solid support. The associated phase changes in the ternary bulk system are accompanied by the formation of a lipid bilayer at the solid-liquid interface. While the phase behavior of the ternary bulk system is well understood, the mechanism of bilayer assembly at the solid-liquid interface remains to be elucidated, including whether the adsorption process is limited by diffusion of the lipid in the bulk or by lipid binding kinetics onto the surface. Such factors strongly influence the success of bilayer formation as they pertain to operating conditions, such as lipid concentration, solvent exchange rate and chamber dimensions, and are hence of critical importance for SALB fabrication strategies. Herein, we extend an earlier proposed phenomenological kinetic model of the SALB formation process, based on a volume-averaged treatment of the solvent mixing process. By comparing the model to quartz crystal microbalance with dissipation monitoring (QCM-D) experimental data, we conclude that SALB formation is limited by diffusion of suspended lipid aggregates, with a hydrodynamic radius, that is consistent with aggregate size measurements in the literature. This agreement validates the proposed model to serve as the basis for optimizing conditions for SALB formation. PMID:27530868

  12. Direct measurement of DNA-mediated adhesion between lipid bilayers.

    PubMed

    Shimobayashi, S F; Mognetti, B M; Parolini, L; Orsi, D; Cicuta, P; Di Michele, L

    2015-06-28

    Multivalent interactions between deformable mesoscopic units are ubiquitous in biology, where membrane macromolecules mediate the interactions between neighbouring living cells and between cells and solid substrates. Lately, analogous artificial materials have been synthesised by functionalising the outer surface of compliant Brownian units, for example emulsion droplets and lipid vesicles, with selective linkers, in particular short DNA sequences. This development extended the range of applicability of DNA as a selective glue, originally applied to solid nano and colloidal particles. On very deformable lipid vesicles, the coupling between statistical effects of multivalent interactions and mechanical deformation of the membranes gives rise to complex emergent behaviours, as we recently contributed to demonstrate [Parolini et al., Nat. Commun., 2015, 6, 5948]. Several aspects of the complex phenomenology observed in these systems still lack a quantitative experimental characterisation and a fundamental understanding. Here we focus on the DNA-mediated multivalent interactions of a single liposome adhering to a flat supported bilayer. This simplified geometry enables the estimate of the membrane tension induced by the DNA-mediated adhesive forces acting on the liposome. Our experimental investigation is completed by morphological measurements and the characterisation of the DNA-melting transition, probed by in situ Förster Resonant Energy Transfer spectroscopy. Experimental results are compared with the predictions of an analytical theory that couples the deformation of the vesicle to a full description of the statistical mechanics of mobile linkers. With at most one fitting parameter, our theory is capable of semi-quantitatively matching experimental data, confirming the quality of the underlying assumptions. PMID:25989828

  13. Examining the origins of the hydration force between lipid bilayers using all-atom simulations.

    PubMed

    Gentilcore, Anastasia N; Michaud-Agrawal, Naveen; Crozier, Paul S; Stevens, Mark J; Woolf, Thomas B

    2010-05-01

    Using 237 all-atom double bilayer simulations, we examined the thermodynamic and structural changes that occur as a phosphatidylcholine lipid bilayer stack is dehydrated. The simulated system represents a micropatch of lipid multilayer systems that are studied experimentally using surface force apparatus, atomic force microscopy and osmotic pressure studies. In these experiments, the hydration level of the system is varied, changing the separation between the bilayers, in order to understand the forces that the bilayers feel as they are brought together. These studies have found a curious, strongly repulsive force when the bilayers are very close to each other, which has been termed the "hydration force," though the origins of this force are not clearly understood. We computationally reproduce this repulsive, relatively free energy change as bilayers come together and make qualitative conclusions as to the enthalpic and entropic origins of the free energy change. This analysis is supported by data showing structural changes in the waters, lipids and salts that have also been seen in experimental work. Increases in solvent ordering as the bilayers are dehydrated are found to be essential in causing the repulsion as the bilayers come together.

  14. Pressure effects on the equilibrium configurations of bilayer lipid membranes

    NASA Astrophysics Data System (ADS)

    DeVita, Raffaella; Stewart, Iain W.; Leo, Donald J.

    2007-10-01

    Planar bilayer lipid membranes (BLMs) are currently employed to construct many bio-inspired material systems and structures. In order to characterize the pressure effects on the equilibrium configurations of these biological membranes, a novel continuum model is proposed. The BLM is assumed to be a two-layer smectic A liquid crystal. The mean orientation of the amphiphilic molecules comprising the membrane is postulated to be perpendicular to the layers and each layer is idealized as a two-dimensional liquid. Moreover, the BLM is modeled as a simply supported plate undergoing small deformations. It is subjected to a pressure load that acts perpendicularly to the layers. The equilibrium equations and boundary conditions are derived from the bulk elastic energy for smectic A liquid crystals as described by de Gennes and Prost (1993 The Physics of Liquid Crystals 2nd edn (Oxford Science Publications)) by using variational methods. The resulting fourth-order linear partial differential equation is solved by employing cylindrical functions and the series solution is proved to be convergent. The solution is numerically computed for values of the model parameters that are reported in the literature. This paper is dedicated to the memory of our colleagues, Professors Kevin P Granata and Liviu Librescv, who lost their lives during the sensless tragedy on 16 April, 2007 at Virginia Tech.

  15. Acyl chain composition and coexisting fluid phases in lipid bilayers

    NASA Astrophysics Data System (ADS)

    Gu, Yongwen; Bradley, Miranda; Mitchell, Drake

    2011-10-01

    At room temperature phospholipid bilayers enriched in sphingolipids and cholesterol may form a solid phase as well as two coexisting fluid phases. These are the standard fluid phase, or the liquid-disordered phase, ld, and the liquid-ordered phase, lo, which is commonly associated with lipid rafts. Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form coexisting lo, ld and solid phases over a wide range of molar ratios. We are examining the ability of two fluorescent probes to detect these 2 phases: NBD linked to di-16:0 PE which partitions strongly into the lo phase and NBD linked to di-18:1 PE which partitions strongly into the ld phase. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the ternary phase diagram of POPC/SPM/Chol with particular focus on the functionally important lo/ld coexistence region. We report on the fluorescence lifetime and anisotropy decay dynamics of these two fluorescent probes.

  16. Millimeter microwave effect on ion transport across lipid bilayer membranes

    SciTech Connect

    Alekseev, S.I.; Ziskin, M.C.

    1995-06-01

    The effects of millimeter microwaves in the frequency range of 54--76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB{sup {minus}}). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1,000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% {+-} 0.5%. At the same time, membrane current induced by TPhB{sup {minus}} transport increased by 5% {+-} 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% {+-} 0.4%). No resonance-like effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB{sup {minus}} transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 C.

  17. Single DNA molecules on freestanding and supported cationic lipid bilayers: diverse conformational dynamics controlled by the local bilayer properties

    NASA Astrophysics Data System (ADS)

    Herold, Christoph; Schwille, Petra; Petrov, Eugene P.

    2016-02-01

    We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid-gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid-gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid-gel coexistence. We also discuss possible biological implications of our experimental findings.

  18. Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

    PubMed Central

    Kalsi, Sumit; Powl, Andrew M.; Wallace, B.A.; Morgan, Hywel; de Planque, Maurits R.R.

    2014-01-01

    Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms. PMID:24739164

  19. Formation of "solvent-free" black lipid bilayer membranes from glyceryl monooleate dispersed in squalene.

    PubMed

    White, S H

    1978-09-01

    A simple technique for forming "black" lipid bilayer membranes containing negligible amounts of alkyl solvent is described. The membranes are formed by the method of Mueller et al (Circulation. 1962. 26:1167.) from glyceryl monooleate (GMO) dispersed in squalene. The squalene forms an annulus to satisfy the boundary conditions of the planar bilayer but does not appear to dissolve noticeably in the bilayer itself. The specific geometric capacitance (Cg) of the membranes at 20 degrees C formed by this technique is 0.7771 +/- 0.0048 muF/cm2. Theoretical estimates of Cg for solvent-free bilayers range from 0.75 to 0.81 muF/cm2. Alkane-free GMO bilayers formed from n-octadecane by the solvent freeze-out method of White (Biochim. Biophys. Acta. 1974. 356:8) have values of Cg = 0.7903 +/- 0.0013 muF/cm2 at 20.5 degrees C. The agreement between the various values of Cg strongly suggests that the bilayers are free of squalene. DC potentials applied to the bilayers have no detectable effect on the value of Cg, as expected for solvent-free films. The ability to form bilayers essentially free of the solvent used in the forming solution makes it possible to determine the area per molecule of the surface active lipid in the bilayer. The area per molecule of GMO at 20 degrees C is estimated to be 37.9 +/- 0.2 A2.

  20. Gramicidin-based fluorescence assay; for determining small molecules potential for modifying lipid bilayer properties.

    PubMed

    Ingólfsson, Helgi I; Sanford, R Lea; Kapoor, Ruchi; Andersen, Olaf S

    2010-10-13

    Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl(+) that has been successfully used in other fluorescence quenching assays. Tl(+) permeates the lipid bilayer slowly but passes readily through conducting gramicidin channels. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results.

  1. Influence of Hydrophobic Mismatch on Structures and Dynamics of Gramicidin A and Lipid Bilayers

    PubMed Central

    Kim, Taehoon; Lee, Kyu Il; Morris, Phillip; Pastor, Richard W.; Andersen, Olaf S.; Im, Wonpil

    2012-01-01

    Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4–5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations—except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch

  2. Influence of hydrophobic mismatch on structures and dynamics of gramicidin a and lipid bilayers.

    PubMed

    Kim, Taehoon; Lee, Kyu Il; Morris, Phillip; Pastor, Richard W; Andersen, Olaf S; Im, Wonpil

    2012-04-01

    Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4-5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations-except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model

  3. High yield formation of lipid bilayer shells around silicon nanowires in aqueous solution.

    PubMed

    Römhildt, Lotta; Gang, Andreas; Baraban, Larysa; Opitz, Jörg; Cuniberti, Gianaurelio

    2013-09-01

    The combination of nanoscaled materials and biological self-assembly is a key step for the development of novel approaches for biotechnology and bionanoelectronic devices. Here we propose a route to merge these two subsystems and report on the formation of highly concentrated aqueous solutions of silanized silicon nanowires wrapped in a lipid bilayer shell. We developed protocols and investigated the dynamics of lipid films on both planar surfaces and silicon nanowires using fluorescence recovery after photobleaching, demonstrating fully intact and fluid bilayers without the presence of a lipid molecule reservoir. Finally, the experimental setup allowed for in situ observation of spontaneous bilayer formation around the nanowire by lipid diffusion from a vesicle to the nanowire. Such aqueous solutions of lipid coated nanowires are a versatile tool for characterization purposes and are relevant for newly emerging bioinspired electronics and nanosensorics.

  4. Regulation of channel function due to physical energetic coupling with a lipid bilayer

    SciTech Connect

    Ashrafuzzaman, Md.; Tseng, C.-Y.; Tuszynski, J.A.

    2014-03-07

    Highlights: • Lipid membrane regulation of membrane protein functions has been addressed. • Energetics behind ion channel-membrane coupling phenomena has been investigated. • Charge based interactions stabilize peptide–lipid complex. • Screened Coulomb interaction model explains the energetics. • Van der Waals and electrostatic forces drive peptides and lipids to close proximity. - Abstract: Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions

  5. Solvent-assisted lipid bilayer formation on silicon dioxide and gold.

    PubMed

    Tabaei, Seyed R; Choi, Jae-Hyeok; Haw Zan, Goh; Zhdanov, Vladimir P; Cho, Nam-Joon

    2014-09-01

    Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance-dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological

  6. MOF nanoparticles coated by lipid bilayers and their uptake by cancer cells.

    PubMed

    Wuttke, Stefan; Braig, Simone; Preiß, Tobias; Zimpel, Andreas; Sicklinger, Johannes; Bellomo, Claudia; Rädler, Joachim O; Vollmar, Angelika M; Bein, Thomas

    2015-11-11

    We report the synthesis of MOF@lipid nanoparticles as a versatile and powerful novel class of nanocarriers based on metal-organic frameworks (MOFs). We show that the MOF@lipid system can effectively store dye molecules inside the porous scaffold of the MOF while the lipid bilayer prevents their premature release. Efficient uptake of the MOF@lipid nanoparticles by cancer cells makes these nanocarriers promising for drug delivery and diagnostic purposes.

  7. Lipid-Bilayer Dynamics Probed by a Carbon Dot-Phospholipid Conjugate.

    PubMed

    Nandi, Sukhendu; Malishev, Ravit; Bhunia, Susanta Kumar; Kolusheva, Sofiya; Jopp, Jürgen; Jelinek, Raz

    2016-05-10

    Elucidating the dynamic properties of membranes is important for understanding fundamental cellular processes and for shedding light on the interactions of proteins, drugs, and viruses with the cell surface. Dynamic studies of lipid bilayers have been constrained, however, by the relatively small number of pertinent molecular probes and the limited physicochemical properties of the probes. We show that a lipid conjugate comprised of a fluorescent carbon dot (C-dot) covalently attached to a phospholipid constitutes a versatile and effective vehicle for studying bilayer dynamics. The C-dot-modified phospholipids readily incorporated within biomimetic membranes, including solid-supported bilayers and small and giant vesicles, and inserted into actual cellular membranes. We employed the C-dot-phospholipid probe to elucidate the effects of polymyxin-B (a cytolytic peptide), valproic acid (a lipophilic drug), and amyloid-β (a peptide associated with Alzheimer's disease) upon bilayer fluidity and lipid dynamics through the application of various biophysical techniques. PMID:27166809

  8. Lipid-Bilayer Dynamics Probed by a Carbon Dot-Phospholipid Conjugate.

    PubMed

    Nandi, Sukhendu; Malishev, Ravit; Bhunia, Susanta Kumar; Kolusheva, Sofiya; Jopp, Jürgen; Jelinek, Raz

    2016-05-10

    Elucidating the dynamic properties of membranes is important for understanding fundamental cellular processes and for shedding light on the interactions of proteins, drugs, and viruses with the cell surface. Dynamic studies of lipid bilayers have been constrained, however, by the relatively small number of pertinent molecular probes and the limited physicochemical properties of the probes. We show that a lipid conjugate comprised of a fluorescent carbon dot (C-dot) covalently attached to a phospholipid constitutes a versatile and effective vehicle for studying bilayer dynamics. The C-dot-modified phospholipids readily incorporated within biomimetic membranes, including solid-supported bilayers and small and giant vesicles, and inserted into actual cellular membranes. We employed the C-dot-phospholipid probe to elucidate the effects of polymyxin-B (a cytolytic peptide), valproic acid (a lipophilic drug), and amyloid-β (a peptide associated with Alzheimer's disease) upon bilayer fluidity and lipid dynamics through the application of various biophysical techniques.

  9. Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels.

    PubMed

    Marin, Victor; Kieffer, Roland; Padmos, Raymond; Aubin-Tam, Marie-Eve

    2016-08-01

    We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

  10. Storable droplet interface lipid bilayers for cell-free ion channel studies.

    PubMed

    Jung, Sung-Ho; Choi, Sangbaek; Kim, Young-Rok; Jeon, Tae-Joon

    2012-01-01

    An artificially created lipid bilayer is an important platform in studying ion channels and engineered biosensor applications. However, a lipid bilayer created using conventional techniques is fragile and short-lived, and the measurement of ion channels requires expertise and laborious procedures, precluding practical applications. Here, we demonstrate a storable droplet lipid bilayer precursor frozen with ion channels, resulting in a droplet interface bilayer upon thawing. A small vial with an aqueous droplet in organic solution was flash frozen in -80 °C methanol immediately after an aqueous droplet was introduced into the organic solution and gravity draws the droplet down to the interface upon thawing. A lipid bilayer created along the interface using this method had giga-ohm resistance and typical specific capacitance values. The noise level of this system is favorably comparable to the conventional system. The subsequent incorporation of ion channels, alpha-hemolysin and gramicidin A, showed typical conductance values consistent with those in previous literatures. This novel system to create a lipid bilayer as a whole can be automated from its manufacture to use and indefinitely stored when frozen. As a result, ion channel measurements can be carried out in any place, increasing the accessibility of ion channel studies as well as a number of applications, such as biosensors, ion channel drug screening, and biophysical studies. PMID:21909672

  11. Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels.

    PubMed

    Marin, Victor; Kieffer, Roland; Padmos, Raymond; Aubin-Tam, Marie-Eve

    2016-08-01

    We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins. PMID:27351219

  12. Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Rønnest, A. K.; Peters, G. H.; Hansen, F. Y.; Taub, H.; Miskowiec, A.

    2016-04-01

    Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic potential within phospholipid membranes imply an enormous electric field of 108-109 V m-1, which is likely to have great significance in controlling the conformation of translocating membrane proteins and in the transfer of ions and molecules across the membrane. We have calculated the membrane potential for DMPG bilayers and found ˜1 V (˜2 ṡ 108 V m-1) when in the fluid phase with a monovalent counter-ion and ˜1.4 V (˜2.8 ṡ 108 V m-1) when in the gel phase with a divalent counter-ion. The number of water molecules for a fully hydrated DMPG membrane has been estimated to be 9.7 molecules per lipid in the gel phase and 17.5 molecules in the fluid phase, considerably smaller than inferred experimentally for 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC

  13. Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size.

    PubMed

    Jing, Yujia; Trefna, Hana; Persson, Mikael; Kasemo, Bengt; Svedhem, Sofia

    2014-01-01

    DPPC liposomes ranging from 90 nm to 160 nm in diameter were prepared and used for studies of the formation of supported lipid membranes on silica (SiO2) at temperatures below and above the gel to liquid-crystalline phase transition temperature (Tm = 41 °C), and by applying temperature gradients through Tm. The main method was the quartz crystal microbalance with dissipation (QCM-D) technique. It was found that liposomes smaller than 100 nm spontaneously rupture on the silica surface when deposited at a temperature above Tm and at a critical surface coverage, following a well-established pathway. In contrast, DPPC liposomes larger than 160 nm do not rupture on the surface when adsorbed at 22 °C or at 50 °C. However, when liposomes of this size are first adsorbed at 22 °C and at a high enough surface coverage, after which they are subject to a constant temperature gradient up to 50 °C, they rupture and fuse to a bilayer, a process that is initiated around Tm. The results are discussed and interpreted considering a combination of effects derived from liposome-surface and liposome-liposome interactions, different softness/stiffness and shape of liposomes below and above Tm, the dynamics and thermal activation of the bilayers occurring around Tm and (for liposomes containing 33% of NaCl) osmotic pressure. These findings are valuable both for preparation of supported lipid bilayer cell membrane mimics and for designing temperature-responsive material coatings. PMID:24651504

  14. Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size.

    PubMed

    Jing, Yujia; Trefna, Hana; Persson, Mikael; Kasemo, Bengt; Svedhem, Sofia

    2014-01-01

    DPPC liposomes ranging from 90 nm to 160 nm in diameter were prepared and used for studies of the formation of supported lipid membranes on silica (SiO2) at temperatures below and above the gel to liquid-crystalline phase transition temperature (Tm = 41 °C), and by applying temperature gradients through Tm. The main method was the quartz crystal microbalance with dissipation (QCM-D) technique. It was found that liposomes smaller than 100 nm spontaneously rupture on the silica surface when deposited at a temperature above Tm and at a critical surface coverage, following a well-established pathway. In contrast, DPPC liposomes larger than 160 nm do not rupture on the surface when adsorbed at 22 °C or at 50 °C. However, when liposomes of this size are first adsorbed at 22 °C and at a high enough surface coverage, after which they are subject to a constant temperature gradient up to 50 °C, they rupture and fuse to a bilayer, a process that is initiated around Tm. The results are discussed and interpreted considering a combination of effects derived from liposome-surface and liposome-liposome interactions, different softness/stiffness and shape of liposomes below and above Tm, the dynamics and thermal activation of the bilayers occurring around Tm and (for liposomes containing 33% of NaCl) osmotic pressure. These findings are valuable both for preparation of supported lipid bilayer cell membrane mimics and for designing temperature-responsive material coatings.

  15. Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

    PubMed

    Wenk, M R; Alt, T; Seelig, A; Seelig, J

    1997-04-01

    The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact

  16. Lateral Pressure Dependence of the Phospholipid Transmembrane Diffusion Rate in Planar-Supported Lipid Bilayers

    PubMed Central

    Anglin, Timothy C.; Conboy, John C.

    2008-01-01

    The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10−4 s−1 to 1.03 × 10−4 s−1) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å2/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed. PMID:18339755

  17. Reduction in lateral lipid mobility of lipid bilayer membrane by atmospheric pressure plasma irradiation

    NASA Astrophysics Data System (ADS)

    Suda, Yoshiyuki; Tero, Ryugo; Yamashita, Ryuma; Yusa, Kota; Takikawa, Hirofumi

    2016-03-01

    Plasma medicine is an emerging research field in which various applications of electrical discharge, especially in the form of nonequilibrium plasma at atmospheric pressure, are examined, for example, the application of plasma to biological targets for various purposes such as selective killing of tumor cells and blood stanching. We have focused on the behavior of an artificial cell membrane system at the solid-liquid interface. To evaluate the lateral lipid mobility, we measured the diffusion coefficient of the supported lipid bilayer (SLB) composed of dioleoylphosphatidylcholine with fluorescence recovery after photobleaching by confocal laser scanning microscopy. It was found that the diffusion coefficient was decreased by plasma irradiation and that the diffusion coefficient decreasing rate proceeded with increasing plasma power. We investigated the effects of stimulation with an equilibrium chemical, H2O2, on the SLB and confirmed that the diffusion coefficient did not change at least up to a H2O2 concentration of 5 mM. These results indicate that transient active species generated by plasma play critical roles in the reduction in SLB fluidity. The effects of the two generated major oxidized lipid species, hydroxyl- or hydroperoxy-phosphatidylcholine (PC) and acyl-chain-truncated PCs terminated with aldehyde or carboxyl group, on lateral lipid mobility are discussed.

  18. Semiconductor particles in bilayer lipid membranes. Formation, characterization, and photoelectrochemistry

    SciTech Connect

    Zhao, X.K.; Baral, S.B.; Rolandi, R.; Fendler, J.H.

    1988-02-17

    Bilayer lipid membranes (BLMs) have been formed from bovine brain phosphatidylserine (PS), glyceryl monooleate (GMO), and a ploymerizable surfactant, (n-C/sub 15/H/sub 31/CO/sub 2/(CH/sub 2/))/sub 2/N/sup +/(CH/sub 3/)CH/sub 2/C/sub 6/H/sub 4/CH==CH/sub 2/Cl/sup -/(STYRS). These BLMs were then used to provide matrices for the in situ generation of microcrystalline CdS, CuS, Cu/sub 2/S, PbS, ZnS, HgS, and In/sub 2/S/sub 3/. Semiconductors were formed by injecting appropriate metal ion precursors and H/sub 2/S into the bathing solutions on opposite sides of the BLM. Their presence was established by voltage-dependent capacitance measurements, absorption spectroscopy, and optical microscopy. Subsequent to the injection of H/sub 2/S, the first observable change was the appearance of fairly uniform white dots on the black film. These dots rapidly moved around and grew in size, forming islands that then merged with themselves and with a second generation of dots, which ultimately led to a continuous film that continued to grow in thickness. Film formation and growth were monitored by simultaneous optical thickness and capacitance measurements. These data were treated in terms of an equivalent R-C circuit and allowed for the assessment of the semiconductor penetration depth into the BLM. This value for a GMO-BLM-supported In/sub 2/S/sub 3/ film was determined to be 24 A. Bandgap excitation, by nanosecond-pulsed or continuous illumination of the BLM-supported semiconductor film, led to observable photoelectric effects. Visible light (lambda > 350 nm) excitation into STYRS-BLM-supported CdS led to polymerization of the styrene moiety of STYRS. BLM-supported semiconductors remained stable for days.

  19. Carrier-mediated ion transport in lipid bilayer membranes.

    PubMed

    Laprade, R; Grenier, F; Pagé-Dansereau, M; Dansereau, J

    1984-08-01

    The electrical properties predicted by a widely accepted model for carrier-mediated ion transport in lipid bilayers are described. The different steps leading to ion transport and their associated rate constants are reaction at the interface between an ion in the aqueous phase and a carrier in the membrane (kRi), followed by translocation of the ion-carrier complex across the membrane interior (kis) and its dissociation at the other interface (kDi) after which the free carrier crosses back the membrane interior (ks). Results on glyceryl monooleate (GMO) membranes for a family of homologue carriers, the macrotetralide actin antibiotics (nonactin, monactin, dinactin, trinactin, and tetranactin) and a variety of ions (Na+, Cs+, Rb+, K+, NH4+, and Tl+) are presented. Internally consistent data obtained from steady-state electrical measurements (zero-current potential and conductance, current-voltage relationship) allow us to obtain the equilibrium permeability ratios for the different ions and show that for a given carrier kRi is relatively invariant from one ion to the other, except for Tl+ (larger), which implies that the ionic selectivity is controlled by the dissociation of the complex. The values of the individual rate constants obtained from current relaxation experiments are also presented and confirm the findings from steady-state measurements, as well as the isostericity concept for complexes of different ions with the same carrier (kis invariant). These also allow us to determine the aqueous phase membrane and torus membrane partition coefficients. Finally, the observed increase in kis from nonactin to tetranactin and, for all homologues, from GMO-decane to solvent-free GMO membranes, together with the concomitant decrease in kDi, can be explained in terms of modifications of electrostatic energy profiles induced by variations in carrier size and membrane thickness.

  20. Charge-transfer processes and redox reactions in planar lipid monolayers and bilayers.

    PubMed

    Krysiñki, P; Tien, H T; Ottova, A

    1999-01-01

    Supported bilayer lipid membranes (BLMs) and lipid monolayers have been known for quite sometime and are attracting sustained interest since they open new research vista and offer practical approaches in biosensor development and molecular device applications. Central to these areas of interest are electric processes and redox reactions where the movement of ions and electrons plays a pivotal role. In this paper an overview of the major findings in this field is presented. Further, we summarize the work on planar lipid bilayers and monolayers that have been done in the past few years in a number of laboratories. Supported planar BLMs and their closely related systems provide the foundation for a variety of lipid bilayer-based molecular sensors that are sensitive, versatile, as well as potentially inexpensive (i.e., disposable), and open to all sorts of experimentation.

  1. Real-time detection of lipid bilayer assembly and detergent-initiated solubilization using optical cavities

    NASA Astrophysics Data System (ADS)

    Sun, V.; Armani, A. M.

    2015-02-01

    The cellular membrane governs numerous fundamental biological processes. Therefore, developing a comprehensive understanding of its structure and function is critical. However, its inherent biological complexity gives rise to numerous inter-dependent physical phenomena. In an attempt to develop a model, two different experimental approaches are being pursued in parallel: performing single cell experiments (top down) and using biomimetic structures (bottom up), such as lipid bilayers. One challenge in many of these experiments is the reliance on fluorescent probes for detection which can create confounds in this already complex system. In the present work, a label-free detection method based on an optical resonant cavity is used to detect one of the fundamental physical phenomena in the system: assembly and solubilization of the lipid bilayer. The evanescent field of the cavity strongly interacts with the lipid bilayer, enabling the detection of the bilayer behavior in real-time. Two independent detection mechanisms confirm the formation and detergent-assisted solubilization of the lipid bilayers: (1) a refractive index change and (2) a material loss change. Both mechanisms can be monitored in parallel, on the same device, thus allowing for cross-confirmation of the results. To verify the proposed method, we have detected the formation of self-assembled phosphatidylcholine lipid bilayers from small unilamellar vesicles on the device surface in real-time. Subsequently, we exposed the bilayers to two different detergents (non-ionic Triton X-100 and anionic sodium dodecyl sulfate) to initiate solubilization, and this process was also detected in real-time. After the bilayer solubilization, the device returned to its initial state, exhibiting minimal hysteresis. The experimental wash-off was also collected and analyzed using dynamic light scattering.

  2. Mechanics of lipid bilayer junctions affecting the size of a connecting lipid nanotube

    NASA Astrophysics Data System (ADS)

    Karlsson, Roger; Kurczy, Michael; Grzhibovskis, Richards; Adams, Kelly L.; Ewing, Andrew G.; Cans, Ann-Sofie; Voinova, Marina V.

    2011-06-01

    In this study we report a physical analysis of the membrane mechanics affecting the size of the highly curved region of a lipid nanotube (LNT) that is either connected between a lipid bilayer vesicle and the tip of a glass microinjection pipette (tube-only) or between a lipid bilayer vesicle and a vesicle that is attached to the tip of a glass microinjection pipette (two-vesicle). For the tube-only configuration (TOC), a micropipette is used to pull a LNT into the interior of a surface-immobilized vesicle, where the length of the tube L is determined by the distance of the micropipette to the vesicle wall. For the two-vesicle configuration (TVC), a small vesicle is inflated at the tip of the micropipette tip and the length of the tube L is in this case determined by the distance between the two interconnected vesicles. An electrochemical method monitoring diffusion of electroactive molecules through the nanotube has been used to determine the radius of the nanotube R as a function of nanotube length L for the two configurations. The data show that the LNT connected in the TVC constricts to a smaller radius in comparison to the tube-only mode and that tube radius shrinks at shorter tube lengths. To explain these electrochemical data, we developed a theoretical model taking into account the free energy of the membrane regions of the vesicles, the LNT and the high curvature junctions. In particular, this model allows us to estimate the surface tension coefficients from R( L) measurements.

  3. Lipid asymmetry in DLPC/DSPC supported lipid bilayers, a combined AFM and fluorescence microscopy study

    SciTech Connect

    Lin, W; Blanchette, C D; Ratto, T V; Longo, M L

    2005-06-20

    A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy (AFM) and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald Ripening, the smaller domains in one leaflet erode away while the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have nearly identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have direct biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

  4. Main phase transitions in supported lipid single-bilayer.

    PubMed

    Charrier, A; Thibaudau, F

    2005-08-01

    We have studied the phase transitions of a phospholipidic single-bilayer supported on a mica substrate by real-time temperature-controlled atomic force microscopy. We show the existence of two phase transitions in this bilayer that we attribute to two gel (L(beta))/fluid (L(alpha)) transitions, corresponding to the independent melting of each leaflet of the bilayer. The ratio of each phase with temperature and the large broadening of the transitions' widths have been interpreted through a basic thermodynamic framework in which the surface tension varies during the transitions. The experimental data can be fit with such a model using known thermodynamic parameters. PMID:15879467

  5. Theory of periodic structures in lipid bilayer membranes

    PubMed Central

    Falkovitz, Meira S.; Seul, Michael; Frisch, Harry L.; McConnell, Harden M.

    1982-01-01

    An approximate, new model for the structure of the periodic, undulated Pβ′, phase of phosphatidylcholine bilayers is proposed. The properties of this phase are deduced by minimizing a Landau-de Gennes expression for the bilayer free energy when this free energy contains a term favoring a spontaneous curvature of the membrane. The theoretical calculation leads to a model for the Pβ′ phase of phosphatidylcholine bilayers having a number of novel physical properties, including periodic variations in membrane “fluidity.” PMID:16593202

  6. Erythrocyte Membrane Model with Explicit Description of the Lipid Bilayer and the Spectrin Network

    PubMed Central

    Li, He; Lykotrafitis, George

    2014-01-01

    The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do

  7. Erythrocyte membrane model with explicit description of the lipid bilayer and the spectrin network.

    PubMed

    Li, He; Lykotrafitis, George

    2014-08-01

    The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do

  8. Electromagnetic and thermal analysis for lipid bilayer membranes exposed to RF fields.

    PubMed

    Eibert, T F; Alaydrus, M; Wilczewski, F; Hansen, V W

    1999-08-01

    Experiments with pulsed radio frequency fields have shown influence on the low-frequency behavior of lipid bilayer membranes. In this paper, we present an electromagnetic and thermal analysis of the used exposure device to clarify whether the observed effects have a thermal cause and to determine the fields at the lipid bilayer. In order to model the very thin lipid bilayer (about 5 nm) accurately, the electromagnetic analysis is broken into several steps employing the finite difference time domain technique and a finite element/boundary element hybrid approach. Based on the obtained power loss due to the electromagnetic fields, the temperature change is calculated using the finite element method for the solution of the heat conduction equation. Both, the electromagnetic and the thermal analysis are performed for a variety of material parameters of the exposure device. The electromagnetic analysis shows that the exposure device is capable of producing voltages on the order of 1 mV across the lipid bilayer. The combined electromagnetic and thermal calculations reveal that the temperature oscillations due to the pulsed radio frequency fields are too small to directly influence the low-frequency behavior of the lipid bilayer.

  9. The Lipid Bilayer Provides a Site for Cortisone Crystallization at High Cortisone Concentrations

    PubMed Central

    Alsop, Richard J.; Khondker, Adree; Hub, Jochen S.; Rheinstädter, Maikel C.

    2016-01-01

    Cortisone is an injected anti-inflammatory drug that can cause painful side effects known as “steroid flares” which are caused by cortisone crystallizing at the injection site. We used molecular dynamics simulations and X-ray diffraction to study the interaction of cortisone with model lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at drug concentrations from 0 mol% to 50 mol%. Cortisone was found to partition in the lipid bilayer and locate in the hydrophilic to hydrophobic interface of the membranes. Cortisone strongly affects the integrity of the membrane, as quantified by a decreased membrane thickness, increased area per lipid, and decreased lipid tail order parameters. At cortisone concentrations of more than 20 mol%, signals from crystallized cortisone were observed. These crystallites are embedded in the bilayers and orient with the membranes. While the cortisone molecules align parallel to the bilayers at low concentrations, they start to penetrate the hydrophobic core at higher concentrations. Trans-membrane crystallites start to nucleate when the membrane thickness has decreased such that cortisone molecules in the different leaflets can find partners from the opposite leaflet resulting in a non-zero density of cortisone molecules in the bilayer center. We suggest that the lipid bilayer provides a site for cortisone crystallization. PMID:26936102

  10. One-Photon Lithography for High-Quality Lipid Bilayer Micropatterns.

    PubMed

    Sánchez, M Florencia; Dodes Traian, Martín M; Levi, Valeria; Carrer, Dolores C

    2015-11-01

    A relevant question in cell biology with broad implications in biomedicine is how the organization and dynamics of interacting membranes modulate signaling cascades that involve cell-cell contact. The functionalization of surfaces with supported lipid bilayers containing tethered proteins is a particularly useful method to present ligands with membrane-like mobility to cells. Here, we present a method to generate micrometer-sized patches of lipid bilayers decorated with proteins. The method uses an economic microcontact printing technique based on one-photon lithography that can be easily implemented in a commercial laser scanning microscope. We verified that both proteins and lipids freely diffuse within the patterned bilayer, as assessed by z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. These results suggest that the supported lipid bilayer patterns constitute an optimal system to explore processes involving direct interactions between cells. We also illustrate possible applications of this method by exploring the interaction of cells expressing the Fas receptor and patterns of lipid bilayers containing an agonist antibody against Fas. PMID:26452154

  11. Disturb or Stabilize? A Molecular Dynamics Study of the Effects of Resorcinolic Lipids on Phospholipid Bilayers

    PubMed Central

    Siwko, Magdalena E.; de Vries, Alex H.; Mark, Alan E.; Kozubek, Arkadiusz; Marrink, Siewert J.

    2009-01-01

    Abstract Resorcinolic lipids, or resorcinols, are commonly found in plant membranes. They consist of a substituted benzene ring forming the hydrophilic lipid head, attached to an alkyl chain forming the hydrophobic tail. Experimental results show alternative effects of resorcinols on lipid membranes. Depending on whether they are added to lipid solutions before or after the formation of the liposomes, they either stabilize or destabilize these liposomes. Here we use atomistic molecular dynamics simulations to elucidate the molecular nature of this dual effect. Systems composed of either one of three resorcinol homologs, differing in the alkyl tail length, interacting with dimyristoylphosphatidylcholine lipid bilayers were studied. It is shown that resorcinols preincorporated into bilayers induce order within the lipid acyl chains, decrease the hydration of the lipid headgroups, and make the bilayers less permeable to water. In contrast, simulations in which the resorcinols are incorporated from the aqueous solution into a preformed phospholipid bilayer induce local disruption, leading to either transient pore formation or even complete rupture of the membrane. In line with the experimental data, our simulations thus demonstrate that resorcinols can either disturb or stabilize the membrane structure, and offer a detailed view of the underlying molecular mechanism. PMID:19383459

  12. Development of an automation technique for the establishment of functional lipid bilayer arrays

    NASA Astrophysics Data System (ADS)

    Hansen, J. S.; Perry, M.; Vogel, J.; Vissing, T.; Hansen, C. R.; Geschke, O.; Emnéus, J.; Nielsen, C. H.

    2009-02-01

    In the present work, a technique for establishing multiple black lipid membranes (BLMs) in arrays of micro structured ethylene tetrafluoroethylene (ETFE) films, and supported by a micro porous material was developed. Rectangular 8 × 8 arrays with apertures having diameters of 301 ± 5 µm were fabricated in ETFE Teflon film by laser ablation using a carbon dioxide laser. Multiple lipid membranes could be formed across the micro structured 8 × 8 array ETFE partitions. Success rates for the establishment of cellulose-supported BLMs across the multiple aperture arrays were above 95%. However, the time course of the membrane thinning process was found to vary considerably between multiple aperture bilayer experiments. An airbrush partition pretreatment technique was developed to increase the reproducibility of the multiple lipid bilayers formation during the time course from the establishment of the lipid membranes to the formation of bilayers. The results showed that multiple lipid bilayers could be reproducible formed across the airbrush-pretreated 8 × 8 rectangular arrays. The ionophoric peptide valinomycin was incorporated into established membrane arrays, resulting in ionic currents that could be effectively blocked by tetraethylammonium. This shows that functional bimolecular lipid membranes were established, and furthermore outlines that the established lipid membrane arrays could host functional membrane-spanning molecules.

  13. A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings.

    PubMed

    Czekalska, Magdalena A; Kaminski, Tomasz S; Jakiela, Slawomir; Tanuj Sapra, K; Bayley, Hagan; Garstecki, Piotr

    2015-01-21

    This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated. PMID:25412368

  14. Bilayer properties of hydroxytyrosol- and tyrosol-phosphatidylcholine lipids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tyrosol and hydroxytyrosol are the phytochemicals abundantly found in olive oil. Transphosphatidylation of tyrosol and hydroxytyrosol with dioleoylphosphocholine resulted in phospholipids with antioxidant properties. The ability of these phyto-phospholipids to form liposomes and supported bilayers w...

  15. Molecular Dynamics of a Water-Lipid Bilayer Interface

    NASA Technical Reports Server (NTRS)

    Wilson, Michael A.; Pohorille, Andrew

    1994-01-01

    We present results of molecular dynamics simulations of a glycerol 1-monooleate bilayer in water. The total length of analyzed trajectories is 5ns. The calculated width of the bilayer agrees well with the experimentally measured value. The interior of the membrane is in a highly disordered fluid state. Atomic density profile, orientational and conformational distribution functions, and order parameters indicate that disorder increases toward the center of the bilayer. Analysis of out-of-plane thermal fluctuations of the bilayer surfaces occurring at the time scale of the present calculations reveals that the distribution of modes agrees with predictions of the capillary wave model. Fluctuations of both bilayer surfaces are uncorrelated, yielding Gaussian distribution of instantaneous widths of the membrane. Fluctuations of the width produce transient thinning defects in the bilayer which occasionally span almost half of the membrane. The leading mechanism of these fluctuations is the orientational and conformational motion of head groups rather than vertical motion of the whole molecules. Water considerably penetrates the head group region of the bilayer but not its hydrocarbon core. The total net excess dipole moment of the interfacial water points toward the aqueous phase, but the water polarization profile is non-monotonic. Both water and head groups significantly contribute to the surface potential across the interface. The calculated sign of the surface potential is in agreement with that from experimental measurements, but the value is markedly overestimated. The structural and electrical properties of the water-bilayer system are discussed in relation to membrane functions, in particular transport of ions and nonelectrolytes across membranes.

  16. Joining patch-clamp and atomic force microscopy techniques for studying black lipid bilayers

    NASA Astrophysics Data System (ADS)

    Ovalle-García, Erasmo; Ortega-Blake, Iván

    2007-08-01

    An experimental protocol that enables the direct characterization of freestanding lipid bilayers through a combination of atomic force microscopy and single channel recording is presented. The method consists of producing a 15μm diameter pore in a 3μm thick Mylar film that delimits two vessels. The micropore was done by a glass microneedle. >L-α-phosphatidylcholine bilayers were routinely painted on the pore, observed by atomic force microscopy, and tested with gramicidin D ion channels.

  17. Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations.

    PubMed

    McIntosh, T J; Kulkarni, K G; Simon, S A

    1999-04-01

    It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion.

  18. Coexistence of a Two-States Organization for a Cell-Penetrating Peptide in Lipid Bilayer

    PubMed Central

    Plénat, Thomas; Boichot, Sylvie; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

    2005-01-01

    Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(α), a primary amphipathic cell-penetrating peptide which remains α-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(α) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(α) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(α). The simultaneous two-states organization of P(α), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide. PMID:16199494

  19. Formation of individual protein channels in lipid bilayers suspended in nanopores.

    PubMed

    Studer, André; Han, Xiaojun; Winkler, Fritz K; Tiefenauer, Louis X

    2009-10-15

    Free-standing lipid bilayers are formed in regularly arranged nanopores of 200, 400 and 800 nm in a 300 nm thin hydrophobic silicon nitride membrane separating two fluid compartments. The extraordinary stability of the lipid bilayers allows us to monitor channel formation of the model peptide melittin and alpha-hemolysin from Staphylococcus aureus using electrochemical impedance spectroscopy and chronoamperometry. We observed that melittin channel formation is voltage-dependent and transient, whereas transmembrane heptameric alpha-hemolysin channels in nano-BLMs persist for hours. The onset of alpha-hemolysin-mediated conduction depends on the applied protein concentration and strongly on the diameter of the nanopores. Heptameric channel formation from adsorbed alpha-hemolysin monomers needs more time in bilayers suspended in 200 nm pores compared to bilayers in pores of 400 and 800 nm diameters. Diffusion of sodium ions across alpha-hemolysin channels present in a sufficiently high number in the bilayers was quantitatively and specifically determined using ion selective electrodes. The results demonstrate that relatively small variations of nano-dimensions have a tremendous effect on observable dynamic biomolecular processes. Such nanopore chips are potentially useful as supports for stable lipid bilayers to establish functional assays of membrane proteins needed in basic research and drug discovery.

  20. Protein stability and conformational rearrangements in lipid bilayers: linear gramicidin, a model system.

    PubMed Central

    Cotten, M; Xu, F; Cross, T A

    1997-01-01

    The replacement of four tryptophans in gramicidin A by four phenylalanines (gramicidin M) causes no change in the molecular fold of this dimeric peptide in a low dielectric isotropic organic solvent, but the molecular folds are dramatically different in a lipid bilayer environment. The indoles of gramicidin A interact with the anisotropic bilayer environment to induce a change in the molecular fold. The double-helical fold of gramicidin M, as opposed to the single-stranded structure of gramicidin A, is not compatible with ion conductance. Gramicidin A/gramicidin M hybrid structures have also been prepared, and like gramicidin M homodimers, these dimeric hybrids appear to have a double-helical fold, suggesting that a couple of indoles are being buried in the bilayer interstices. To achieve this equilibrium structure (i.e., minimum energy conformation), incubation at 68 degrees C for 2 days is required. Kinetically trapped metastable structures may be more common in lipid bilayers than in an aqueous isotropic environment. Structural characterizations in the bilayers were achieved with solid-state NMR-derived orientational constraints from uniformly aligned lipid bilayer samples, and characterizations in organic solvents were accomplished by solution NMR. Images FIGURE 2 FIGURE 3 FIGURE 5 PMID:9251781

  1. Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts.

    PubMed

    Marquês, Joaquim T; Viana, Ana S; De Almeida, Rodrigo F M

    2011-01-01

    Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization. PMID:20955684

  2. Modeling analysis of the lipid bilayer-cytoskeleton coupling in erythrocyte membrane.

    PubMed

    Pajic-Lijakovic, Ivana; Milivojevic, Milan

    2014-10-01

    Studies of thermal fluctuations in discocytes, echinocytes, and spherocytes suggest that the coupling between lipid bilayer and cytoskeleton can affect viscoelastic behavior of single erythrocyte membranes. To test this hypothesis, we developed a 3D constitutive model describing viscoelastic behavior of erythrocyte membranes, at long relaxation times [Formula: see text] and short relaxation times [Formula: see text]. The model was constructed using combination of spring and spring pot rheological elements arranged in parallel. The rearrangement of cytoskeleton induced by changing the bending state of lipid bilayer was described by a modified Eyring model. The model predictions point to an anomalous nature of energy dissipation and an ordered harmonic nature of the coupling mechanism described by a series of fractional derivatives of the order n [Formula: see text] (where [Formula: see text]). As a result, the stress generated within the lipid bilayer is related to the rate of change of the irreversible stress within the cytoskeleton.

  3. Two states of cyclic antimicrobial peptide RTD-1 in lipid bilayers.

    PubMed

    Weiss, Thomas M; Yang, Lin; Ding, Lai; Waring, Alan J; Lehrer, Robert I; Huang, Huey W

    2002-08-01

    RTD-1 is a recently discovered cyclic peptide that, like other well-studied antimicrobial peptides, appears to bind to the lipid matrix of cell membrane in the initial stage of activity. We studied the states of RTD-1 bound to lipid bilayers by two methods: oriented circular dichroism and X-ray diffraction. RTD-1 shows two physically distinct bound states in lipid bilayers like magainins, protegrins, alamethicin, and melittin that were previously studied. However, the nature of transition between the two states is different for RTD-1 as compared with the aforementioned peptides. In one of the two states, RTD-1 is oriented with its backbone ring parallel to the plane of the bilayer. Only in this state RTD-1 induces membrane thinning. But the effect of membrane thinning is much weaker than all other peptides, suggesting that the mechanism of RTD-1 may be different from the other peptides. PMID:12146971

  4. Planar lipid bilayers containing gramicidin A as a molecular sensing system based on an integrated current.

    PubMed

    Nishio, Masato; Shoji, Atsushi; Sugawara, Masao

    2012-01-01

    The channel activity of gramicidin A in free-standing planar lipid bilayers with different charges of polar head groups and various lengths of hydrocarbon tails were analyzed in terms of the channel conductance, the lifetime of channel events and the magnitude of integrated currents. The channel activity of gramicidin A in lipid bilayers is tunable by adjusting the membrane composition. The in situ coupling of the anti-BSA antibody as a model protein to the amine moiety of phosphatidylethanolamine (PE) in a lipid bilayer by the amine coupling method allowed us to design an antigen (BSA)-sensitive interface, in which the integrated current, rather than the frequency of channel event, can be used as an analytical signal. The potential of the present system for highly sensitive and selective detection of BSA at 10(-9) g/mL level is demonstrated.

  5. Lipid clustering in bilayers detected by the fluorescence kinetics and anisotropy of trans-parinaric acid.

    PubMed Central

    Reyes Mateo, C; Brochon, J C; Pilar Lillo, M; Ulises Acuña, A

    1993-01-01

    Fluid heterogeneity in lipid bilayers and shows a simple and useful method to quantify this heterogeneity. Taking advantage of the maximum entropy method, we have resolved the probe fluorescence lifetime distributions in homogeneous solutions and in single and two-component lipid bilayers at different temperatures. A precise description of the emission kinetics was obtained as a function of viscosity in the homogeneous solution and as a function of the phase composition (gel/fluid) in the lipid bilayers. These data show, unambiguously, that the same distribution pattern, with two well resolved lifetime classes, is observed both in pure solvents and in fluid bilayers. This distribution is modified during the thermotropic phase transition, with the appearance of a long lifetime component. The anisotropy experiments confirm that the amplitude of this component is proportional to the fraction of probe located in the gel phase. From this fraction we have quantified the amount of gel phase in the binary bilayer system dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine and determined the thermotropic phase diagram of the mixture. This phase diagram agrees well with that calculated assuming ideal mixing of the lipids (Marbrey, S., and J.M. Sturtevant. 1976. Proc. Natl. Acad. Sci. USA. 73:862-3866). PMID:8298047

  6. Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

    SciTech Connect

    Drücker, Patrick; Gerke, Volker; Galla, Hans-Joachim

    2014-10-10

    Highlights: • We show long-term mechanical stabilization of solid supported bilayers. • Bilayer integrity is essential for the investigation of protein–lipid interactions. • Protein adsorption to a bilayer containing defects causes membrane destruction. - Abstract: The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO{sub 2} and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P{sub 2} (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P{sub 2} and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.

  7. Ion channel stability of Gramicidin A in lipid bilayers: effect of hydrophobic mismatch.

    PubMed

    Basu, Ipsita; Chattopadhyay, Amitabha; Mukhopadhyay, Chaitali

    2014-01-01

    Hydrophobic mismatch which is defined as the difference between the lipid hydrophobic thickness and the peptide hydrophobic length is known to be responsible in altering the lipid/protein dynamics. Gramicidin A (gA), a 15 residue β helical peptide which is well recognized to form ion conducting channels in lipid bilayer, may change its structure and function in a hydrophobic mismatched condition. We have performed molecular dynamics simulations of gA dimer in phospholipid bilayers to investigate whether or not the conversion from channel to non-channel form of gA dimer would occur under extreme negative hydrophobic mismatch. By varying the length of lipid bilayers from DLPC (1, 2-Dilauroyl-sn-glycero-3-phosphocholine) to DAPC (1, 2-Diarachidoyl-sn-glycero-3-phosphocholine), a broad range of mismatch was considered from nearly matching to extremely negative. Our simulations revealed that though the ion-channel conformation is retained by gA under a lesser mismatched situation, in extremely negative mismatched situation, in addition to bilayer thinning, the conformation of gA is changed and converted to a non-channel one. Our results demonstrate that although the channel conformation of Gramicidin A is the most stable structure, it is possible for gA to change its conformation from channel to non-channel depending upon the local environment of host bilayers.

  8. Morphological and nanomechanical behavior of supported lipid bilayers on addition of cationic surfactants.

    PubMed

    Lima, Lia M C; Giannotti, Marina I; Redondo-Morata, Lorena; Vale, M Luísa C; Marques, Eduardo F; Sanz, Fausto

    2013-07-30

    The addition of surfactants to lipid bilayers is important for the modulation of lipid bilayer properties (e.g., in protein reconstitution and development of nonviral gene delivery vehicles) and to provide insight on the properties of natural biomembranes. In this work, the thermal behavior, organization, and nanomechanical stability of model cationic lipid-surfactant bilayers have been investigated. Two different cationic surfactants, hexadecyltrimethylammonium bromide (CTAB) and a novel derivative of the amino acid serine (Ser16TFAc), have been added (up to 50 mol %) to both liposomes and supported lipid bilayers (SLBs) composed by the zwitterionic phospholipid DPPC. The thermal phase behavior of mixed liposomes has been probed by differential scanning calorimetry (DSC), and the morphology and nanomechanical properties of mixed SLBs by atomic force microscopy-based force spectroscopy (AFM-FS). Although DSC thermograms show different results for the two mixed liposomes, when both are deposited on mica substrates similar trends on the morphology and the mechanical response of the lipid-surfactant bilayers are observed. DSC thermograms indicate microdomain formation in both systems, but while CTAB decreases the degree of organization on the liposome bilayer, Ser16TFAc ultimately induces the opposite effect. Regarding the AFM-FS studies, they show that microphase segregation occurs for these systems and that the effect is dependent on the surfactant content. In both SLB systems, different microdomains characterized by their height and breakthrough force Fb are formed. The molecular organization and composition is critically discussed in the light of our experimental results and literature data on similar lipid-surfactant systems. PMID:23782267

  9. Insulin-induced changes in mechanical characteristics of lipid bilayers modified by liver plasma membrane fragments.

    PubMed

    Hianik, T; Kavecanský, J; Zórad, S; Macho, L

    1988-04-01

    Insulin interaction with BLM with incorporated fragments of rat liver plasma membranes, containing hormone receptors, was studied by determining Young modulus of elasticity of bilayer lipid membranes in direction perpendicular to the surface, E. The presence of membrane proteins in a concentration of 60 micrograms.ml-1 induced a significant decrease in parameter E (to approx. 50%) as compared with values obtained in non-modified membranes during insulin action (concentration interval 10(-11)-10(-9) mol.l-1). The extent of the effect was dependent on the initial phase state of the membrane, on cholesterol content in BLM as well as on membrane proteins concentration in lipid bilayer.

  10. Sampling errors in free energy simulations of small molecules in lipid bilayers.

    PubMed

    Neale, Chris; Pomès, Régis

    2016-10-01

    Free energy simulations are a powerful tool for evaluating the interactions of molecular solutes with lipid bilayers as mimetics of cellular membranes. However, these simulations are frequently hindered by systematic sampling errors. This review highlights recent progress in computing free energy profiles for inserting molecular solutes into lipid bilayers. Particular emphasis is placed on a systematic analysis of the free energy profiles, identifying the sources of sampling errors that reduce computational efficiency, and highlighting methodological advances that may alleviate sampling deficiencies. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

  11. The mechanism of detergent solubilization of lipid bilayers.

    PubMed

    Lichtenberg, Dov; Ahyayauch, Hasna; Goñi, Félix M

    2013-07-16

    Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted detergents. The structure of such mixed assemblies has been thoroughly investigated, described in phase diagrams, and theoretically rationalized in terms of the balance between the large spontaneous curvature of the curvophilic detergent and the curvophobic phospholipids. In this critical review, we discuss the mechanism of this process and try to explain the actual mechanism involved in solubilization. Interestingly, membrane solubilization by some detergents is relatively slow and the common attribute of these detergents is that their trans-bilayer movement, commonly denoted flip-flop, is very slow. Only detergents that can flip into the inner monolayer cause relatively rapid solubilization of detergent-saturated bilayers. This occurs via the following sequence of events: 1), relatively rapid penetration of detergent monomers into the outer monolayer; 2), trans-membrane equilibration of detergent monomers between the two monolayers; 3), saturation of the bilayer by detergents and consequent permeabilization of the membrane; and 4), transition of the whole bilayer to thread-like mixed micelles. When the detergent cannot flip to the inner monolayer, the outer monolayer becomes unstable due to mass imbalance between the monolayers and inclusion of the curvophilic detergent molecules in a flat surface. Consequently, the outer monolayer forms mixed micellar structures within the outer monolayer. Shedding of these micelles into the aqueous solution results in partial solubilization. The consequent leakage of detergent into the liposome results in trans-membrane equilibration of detergent and subsequent micellization through the rapid bilayer-saturation mechanism.

  12. Interphases of chain molecules: Monolayers and lipid bilayer membranes

    PubMed Central

    Dill, Ken A.; Flory, Paul J.

    1980-01-01

    Using the lattice model for a liquid, we treat the packing of short-chain molecules in interphases such as bilayer membranes. The constant density in the interphase imposes intermolecular constraints on the configurations of the flexible chains. The statistical theory here presented predicts a diffuse distribution of chain ends near the bilayer midplane; no adjustable parameters are required. Inasmuch as some of the chains terminate relatively near the polar interface, the number of chains reaching deeper planar layers is diminished. Consequently, configurational freedom increases with depth. This is the source of the well-known disorder gradient. PMID:16592834

  13. Teflon™-coated silicon apertures for supported lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Wilk, S. J.; Goryll, M.; Laws, G. M.; Goodnick, S. M.; Thornton, T. J.; Saraniti, M.; Tang, J.; Eisenberg, R. S.

    2004-10-01

    We present a method for microfabricating apertures in a silicon substrate using well-known cleanroom technologies resulting in highly reproducible giga-seal resistance bilayer formations. Using a plasma etcher, 150μm apertures have been etched through a silicon wafer. Teflon™ has been chemically vapor deposited so that the surface resembles bulk Teflon and is hydrophobic. After fabrication, reproducible high resistance bilayers were formed and characteristic measurements of a self-inserted single OmpF porin ion channel protein were made.

  14. Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

    PubMed Central

    Heyse, S.; Vogel, H.; Sänger, M.; Sigrist, H.

    1995-01-01

    A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde

  15. Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

    PubMed

    Hare, Joanna E; Goodchild, Sophia C; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

    2016-07-12

    Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35. PMID:27299171

  16. Fluid Phase Lipid Areas and Bilayer Thicknesses of Commonly Used Phosphatidylcholines as a Function of Temperature

    SciTech Connect

    Kucerka, Norbert; Nieh, Mu-Ping; Katsaras, John

    2011-01-01

    The structural parameters of fluid phase bilayers composed of phosphatidylcholines with fully saturated, mixed, and branched fatty acid chains, at several temperatures, have been determined by simultaneously analyzing small-angle neutron and X-ray scattering data. Bilayer parameters, such as area per lipid and overall bilayer thickness have been obtained in conjunction with intrabilayer structural parameters (e.g. hydrocarbon region thickness). The results have allowed us to assess the effect of temperature and hydrocarbon chain composition on bilayer structure. For example, we found that for all lipids there is, not surprisingly, an increase in fatty acid chain trans-gauche isomerization with increasing temperature. Moreover, this increase in trans-gauche isomerization scales with fatty acid chain length in mixed chain lipids. However, in the case of lipids with saturated fatty acid chains, trans-gauche isomerization is increasingly tempered by attractive chain-chain van der Waals interactions with increasing chain length. Finally, our results confirm a strong dependence of lipid chain dynamics as a function of double bond position along fatty acid chains.

  17. Probe chemistry effect on surface properties of asymmetric-phase lipid bilayers.

    PubMed

    Park, Jin-Won

    2010-01-01

    Phospholipid bilayers were formed on mica using Langmuir-Blodgett technique and liposome fusion, as a model system for biomembranes. Nanometer-scale surface physical properties were quantitatively characterized upon the different phases of the monolayers with the different surface chemistry. The less hydration/steric forces were observed at the liquid-phase of the lipid layer than at the solid-phase for the OH-modified probe, while the forces with the CH(3)-modified probe were independent of the mechanical stability of the layer. The forces appear to be related to the surface chemistry of the probe to the layer as well as the mechanical stability of the lipid layer, which depends on the phase and the asymmetry of the lipid bilayer. After the breakthrough of the lipid bilayer, the CH(3)-modified probe adhered more strongly to the lipid bilayers than do the OH-modified probe. Using results from the JKR theory, it is found that the adhesion can be accounted for in both cases by surface energy consideration, not mechanical effects.

  18. Charged particles interacting with a mixed supported lipid bilayer as a biomimetic pulmonary surfactant.

    PubMed

    Munteanu, B; Harb, F; Rieu, J P; Berthier, Y; Tinland, B; Trunfio-Sfarghiu, A-M

    2014-08-01

    This study shows the interactions of charged particles with mixed supported lipid bilayers (SLB) as biomimetic pulmonary surfactants. We tested two types of charged particles: positively charged and negatively charged particles. Two parameters were measured: adsorption density of particles on the SLB and the diffusion coefficient of lipids by FRAPP techniques as a measure of interaction strength between particles and lipids. We found that positively charged particles do not adsorb on the bilayer, probably due to the electrostatic repulsion between positively charged parts of the lipid head and the positive groups on the particle surface, therefore no variation in diffusion coefficient of lipid molecules was observed. On the contrary, the negatively charged particles, driven by electrostatic interactions are adsorbed onto the supported bilayer. The adsorption of negatively charged particles increases with the zeta-potential of the particle. Consecutively, the diffusion coefficient of lipids is reduced probably due to binding onto the lipid heads which slows down their Brownian motion. The results are directly relevant for understanding the interactions of particulate matter with pulmonary structures which could lead to pulmonary surfactant inhibition or deficiency causing severe respiratory distress or pathologies.

  19. Swing motion as a diffusion mechanism of lipid bilayers in a gel phase.

    PubMed

    Oh, Younghoon; Kim, Jeongmin; Yethiraj, Arun; Sung, Bong June

    2016-01-01

    Lipid bilayers are a model system for studying the properties of cell membranes. For lipid bilayers of a single lipid component, there is a phase transition from a fluid phase to a gel phase as the temperature is decreased. The dynamic behavior of lipids in the gel phase is interesting: some models show dynamic heterogeneity with a large disparity in timescales between fast and slow molecules, and a spatial segregation of the slow molecules. In this paper we study the dynamics of coarse-grained models of lipid bilayers using the dry Martini, Lennard-Jones Martini, polarizable Martini, and BMW models. All four models show similar dynamical behaviors in the gel phase although the transition temperature is model-dependent. We find that the primary mode of transport in the gel phase is a hopping of the lipid molecules. Hopping is seen in both the translational and rotational dynamics, which are correlated, i.e., the lipid molecules display a swing-like motion in the gel phase. PMID:26871103

  20. Swing motion as a diffusion mechanism of lipid bilayers in a gel phase

    NASA Astrophysics Data System (ADS)

    Oh, Younghoon; Kim, Jeongmin; Yethiraj, Arun; Sung, Bong June

    2016-01-01

    Lipid bilayers are a model system for studying the properties of cell membranes. For lipid bilayers of a single lipid component, there is a phase transition from a fluid phase to a gel phase as the temperature is decreased. The dynamic behavior of lipids in the gel phase is interesting: some models show dynamic heterogeneity with a large disparity in timescales between fast and slow molecules, and a spatial segregation of the slow molecules. In this paper we study the dynamics of coarse-grained models of lipid bilayers using the dry Martini, Lennard-Jones Martini, polarizable Martini, and BMW models. All four models show similar dynamical behaviors in the gel phase although the transition temperature is model-dependent. We find that the primary mode of transport in the gel phase is a hopping of the lipid molecules. Hopping is seen in both the translational and rotational dynamics, which are correlated, i.e., the lipid molecules display a swing-like motion in the gel phase.

  1. Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study.

    PubMed

    Sacchi, Mattia; Balleza, Daniel; Vena, Giulia; Puia, Giulia; Facci, Paolo; Alessandrini, Andrea

    2015-05-01

    Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

  2. A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress.

    PubMed

    Vyšniauskas, Aurimas; Qurashi, Maryam; Kuimova, Marina K

    2016-09-01

    Oxidation of cellular structures is typically an undesirable process that can be a hallmark of certain diseases. On the other hand, photooxidation is a necessary step of photodynamic therapy (PDT), a cancer treatment causing cell death upon light irradiation. Here, the effect of photooxidation on the microscopic viscosity of model lipid bilayers constructed of 1,2-dioleoyl-sn-glycero-3-phosphocholine has been studied. A molecular rotor has been employed that displays a viscosity-dependent fluorescence lifetime as a quantitative probe of the bilayer's viscosity. Thus, spatially-resolved viscosity maps of lipid photooxidation in giant unilamellar vesicles (GUVs) were obtained, testing the effect of the positioning of the oxidant relative to the rotor in the bilayer. It was found that PDT has a strong impact on viscoelastic properties of lipid bilayers, which 'travels' through the bilayer to areas that have not been irradiated directly. A dramatic difference in viscoelastic properties of oxidized GUVs by Type I (electron transfer) and Type II (singlet oxygen-based) photosensitisers was also detected. PMID:27487026

  3. Stabilising lamellar stacks of lipid bilayers with soft confinement and steric effects.

    PubMed

    Bougis, K; Leite Rubim, R; Ziane, N; Peyencet, J; Bentaleb, A; Février, A; Oliveira, C L P; Andreoli de Oliveira, E; Navailles, L; Nallet, F

    2015-07-01

    Structure and interactions stabilising the lamellar stack of mixed lipid bilayers in their fluid state are investigated by means of small-angle X-ray scattering. The (electrically neutral) bilayers are composed of a mixtures of lecithin, a zwitterionic phospholipid, and Simulsol, a non-ionic cosurfactant with an ethoxylated polar head. The soft confinement of the bilayer hydrophilic components is varied by changing hydration and bilayer composition, as well as the length of the cosurfactant polar head. Structural transitions are observed at low hydration, in the stacking order for the longer cosurfactant, and in the mixed bilayers for the shorter one. At higher hydration, the swelling of the lamellar stacks occurs with a significant, but continuous evolution in the mixed bilayer structure. The bilayer structural changes are discussed in analogy with the so-called "brush-to-mushroom" transition induced by lateral confinement, relevant for long linear polymers grafted onto rigid surfaces, taking also into account the role of vertical confinement.

  4. Structural Degradation and Swelling of Lipid Bilayer under the Action of Benzene.

    PubMed

    Odinokov, Alexey; Ostroumov, Denis

    2015-12-01

    Benzene and other nonpolar organic solvents can accumulate in the lipid bilayer of cellular membranes. Their effect on the membrane structure and fluidity determines their toxic properties and antibiotic action of the organic solvents on the bacteria. We performed molecular dynamics simulations of the interaction of benzene with the dimyristoylphosphatidylcholine (DMPC) bilayer. An increase in the membrane surface area and fluidity was clearly detected. Changes in the acyl chain ordering, tilt angle, and overall bilayer thickness were, however, much less marked. The dependence of all computed quantities on the benzene content showed two regimes separated by the solubility limit of benzene in water. When the amount of benzene exceeded this point, a layer of almost pure benzene started to grow between the membrane leaflets. This process corresponds to the nucleation of a new phase and provides a molecular mechanism for the mechanical rupture of the bilayer under the action of nonpolar compounds.

  5. Capsaicin interaction with TRPV1 channels in a lipid bilayer: molecular dynamics simulation.

    PubMed

    Hanson, Sonya M; Newstead, Simon; Swartz, Kenton J; Sansom, Mark S P

    2015-03-24

    Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1-S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1-S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1-S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular

  6. The local heating effect by magnetic nanoparticles aggregate on support lipid bilayers.

    PubMed

    Wang, Changling; Xu, Ruizhi; Tang, Liming

    2013-07-01

    In this paper, we established a theoretical model to investigate the local heating effect of magnetic nanoparticles (MNPs) aggregate on the support lipid bilayers (SLBs) under external alternating current (AC) magnetic field, which may be helpful to understand hyperthermia at single cell level. Using atomic force microscope (AFM), the transformation of the support phospholipid bilayers surrounding MNPs aggregate was observed in real-time. We found that the fluidity of lipid bilayers changed when the size of MNPs aggregate larger than 200 nm, as a result of magnetic heating in the AC magnetic field. These experimental data were consistent with the simulation results, which demonstrated the valid of our established model, as well as described more realistically the above phenomenon.

  7. Construction and characterization of soft-supported lipid bilayer membranes for biosensors application.

    PubMed

    Jimenez, Jeffy; Heim, August J; Matthews, W Garrett; Alcantar, Norma

    2006-01-01

    A model cell membrane can be ingeniously used to mimic biological processes with wide applications in the biomedical field. For instance, a model cell membrane can be used as a functionalized element integrated to a sensor for identification of specific proteins or other molecules in a biofluid. A special characteristic of the model membrane described in this work is the fluidic or surface mobility feature given by a thin film of grafted polymer that serves as a cushion layer to support lipid bilayers. Lipid bilayers were deposited on a polyethylene glycol (PEG) thin film over a polished glass substrate using the Langmuir-Blodgett technique. Topographical characterization of the bilayers was accomplished using atomic force microscopy (AFM). Results revealed that the grafted polymeric PEG cushion layer confers mobility to the model membrane. PMID:17947068

  8. Interactions of ibuprofen with hybrid lipid bilayers probed by complementary surface-enhanced vibrational spectroscopies

    PubMed Central

    Levin, Carly S.; Kundu, Janardan; Janesko, Benjamin G.; Scuseria, Gustavo E.; Raphael, Robert M.; Halas, Naomi J.

    2016-01-01

    The incorporation of small molecules into lipid bilayers is a process of biological importance and clinical relevance that can change the material properties of cell membranes and cause deleterious side effects for certain drugs. Here we report the direct observation, using surface enhanced Raman and IR spectroscopies (SERS, SEIRA), of the insertion of ibuprofen molecules into hybrid lipid bilayers. The alkanethiol-phospholipid hybrid bilayers were formed onto gold nanoshells by self-assembly, where the underlying nanoshell substrates provided the necessary enhancements for SERS and SEIRA. The spectroscopic data reveal specific interactions between ibuprofen and phospholipid moieties and indicate that the overall hydrophobicity of ibuprofen plays an important role in its intercalation in these membrane mimics. PMID:18942873

  9. Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

    NASA Astrophysics Data System (ADS)

    Suzuki, Yuki; Endo, Masayuki; Sugiyama, Hiroshi

    2015-08-01

    Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed `lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space.

  10. Insertion mechanism and stability of boron nitride nanotubes in lipid bilayers.

    PubMed

    Thomas, Michael; Enciso, Marta; Hilder, Tamsyn A

    2015-04-16

    We provide insight into the interaction of boron nitride nanotubes (BNNTs) with cell membranes to better understand their improved biocompatibility compared to carbon nanotubes (CNTs). Contrary to CNTs, no computational studies exist investigating the insertion mechanism and stability of BNNTs in membranes. Our molecular dynamics simulations demonstrate that BNNTs are spontaneously attracted to lipid bilayers and are stable once inserted. They insert via a lipid-mediated, passive insertion mechanism. BNNTs demonstrate similar characteristics to more biocompatible functionalized CNTs.

  11. One-dimensional lipid bilayers on carbon nanotubes: Structure and properties

    NASA Astrophysics Data System (ADS)

    Artyukhin, Alexander Borisovich

    In this work we report design, assembly, and properties of one-dimensional lipid bilayers wrapped around polymer-coated carbon nanotubes. We propose to use this platform as a tool for interfacing nanomaterials with biological systems. We start by presenting a new general procedure for noncovalent modification of carbon nanotubes based on polyelectrolyte layer-by-layer assembly. We confirm formation of multilayer polymer structures around individual carbon nanotubes by transmission electron microscopy and confocal fluorescence microscopy, and demonstrate that sign of the outmost polymer layer controls surface properties of the multilayer assembly. We study how rigidity of a polymer chain influences its ability to adsorb onto high curvature substrates, such as carbon nanotubes. We then build the one-dimensional lipid bilayer structure by spontaneous assembly of lipid molecules in a continuous nanoshell around a template of a carbon nanotube wrapped with hydrophilic polymer cushion layers. We demonstrate that such one-dimensional lipid membranes are fluid and can heal defects, even over repeated damage-recovery cycles. Measured diffusion coefficients of lipid molecules in our polymer-supported bilayers are about 3 orders of magnitude lower than typical values for fluid lipid membranes, which we attribute to strong electrostatic polyelectrolyte-lipid interactions. To explore the potential for device integration of one-dimensional bilayers we investigate effect of polyelectrolyte multilayers on electrical properties of carbon nanotube transistors. We demonstrate that complex interaction of adsorbed species with the device substrate can produce significant and sometimes unexpected side effects on device characteristics. Finally, we fabricate transistors with suspended carbon nanotube channels and devise a method to transfer them in liquid. It allows us to assemble one-dimensional lipid membranes on carbon nanotube devices, characterize their electrical properties, and

  12. Self-consistent mean-field model for palmitoyloleoylphosphatidylcholine-palmitoyl sphingomyelin–cholesterol lipid bilayers

    PubMed Central

    Tumaneng, Paul W.; Pandit, Sagar A.; Zhao, Guijun; Scott, H. L.

    2012-01-01

    The connection between membrane inhomogeneity and the structural basis of lipid rafts has sparked interest in the lateral organization of model lipid bilayers of two and three components. In an effort to investigate anisotropic lipid distribution in mixed bilayers, a self-consistent mean-field theoretical model is applied to palmitoyloleoylphosphatidylcholine (POPC)–palmitoyl sphingomyelin (PSM)–cholesterol mixtures. The compositional dependence of lateral organization in these mixtures is mapped onto a ternary plot. The model utilizes molecular dynamics simulations to estimate interaction parameters and to construct chain conformation libraries. We find that at some concentration ratios the bilayers separate spatially into regions of higher and lower chain order coinciding with areas enriched with PSM and POPC, respectively. To examine the effect of the asymmetric chain structure of POPC on bilayer lateral inhomogeneity, we consider POPC-lipid interactions with and without angular dependence. Results are compared with experimental data and with results from a similar model for mixtures of dioleoylphosphatidylcholine, steroyl sphingomyelin, and cholesterol. PMID:21517541

  13. Self-consistent mean-field model for palmitoyloleoylphosphatidylcholine-palmitoyl sphingomyelin-cholesterol lipid bilayers

    NASA Astrophysics Data System (ADS)

    Tumaneng, Paul W.; Pandit, Sagar A.; Zhao, Guijun; Scott, H. L.

    2011-03-01

    The connection between membrane inhomogeneity and the structural basis of lipid rafts has sparked interest in the lateral organization of model lipid bilayers of two and three components. In an effort to investigate anisotropic lipid distribution in mixed bilayers, a self-consistent mean-field theoretical model is applied to palmitoyloleoylphosphatidylcholine (POPC)-palmitoyl sphingomyelin (PSM)-cholesterol mixtures. The compositional dependence of lateral organization in these mixtures is mapped onto a ternary plot. The model utilizes molecular dynamics simulations to estimate interaction parameters and to construct chain conformation libraries. We find that at some concentration ratios the bilayers separate spatially into regions of higher and lower chain order coinciding with areas enriched with PSM and POPC, respectively. To examine the effect of the asymmetric chain structure of POPC on bilayer lateral inhomogeneity, we consider POPC-lipid interactions with and without angular dependence. Results are compared with experimental data and with results from a similar model for mixtures of dioleoylphosphatidylcholine, steroyl sphingomyelin, and cholesterol.

  14. Thermal fluctuations in shape, thickness, and molecular orientation in lipid bilayers

    NASA Astrophysics Data System (ADS)

    Watson, Max C.; Penev, Evgeni S.; Welch, Paul M.; Brown, Frank L. H.

    2011-12-01

    We present a unified continuum-level model for bilayer energetics that includes the effects of bending, compression, lipid orientation (tilting relative to the monolayer surface normal), and microscopic noise (protrusions). Expressions for thermal fluctuation amplitudes of several physical quantities are derived. These predictions are shown to be in good agreement with molecular simulations.

  15. Mesoscopic Simulations of the Insertion of the Amphiphilic Nanotubes into Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Dutt, Meenakshi; Nayhouse, Michael; Kuksenok, Olga; Alexeev, Alexander; Little, Steven R.; Balazs, Anna C.

    2010-03-01

    Using Dissipative Particle Dynamics (DPD) simulations, we investigate the interactions between amphiphilic nanotubes and a lipid bilayer membrane. Each nanotube encompasses an ABA triblock (TB) architecture, with a hydrophobic stalk and two hydrophilic ends. To allow controlled transport through the nanotube, we introduce hydrophilic tethers at one or both ends of the nanotube. Individual lipids are composed of a hydrophilic head group and two hydrophobic tails. We begin with a stable lipid bilayer membrane immersed in a hydrophilic solvent, and introduce the nanotube into the surrounding solution. The energetically unfavorable interaction between the solvent and the hydrophobic segment of the nanotube could potentially drive them to penetrate the membrane, with the hydrophobic stalk being buried within the hydrophobic domains of the bilayer. This process, however, depends upon the hydrophobic fraction of the nanotube, the degree of hydrophobic mismatch between the nanotube and the bilayer, and the presence of hydrophilic end-tethers. We isolate the conditions that promote the insertion of the synthetic nanotubes into membrane. The simulations are supported by free energy calculations for the amphiphilic nanotube-lipid-solvent system. Ultimately, these embedded synthetic nanotubes could be used to regulate the passage of molecules through synthetic membranes.

  16. Location, Partitioning Behavior, and Interaction of Capsaicin with Lipid Bilayer Membrane: Study Using Its Intrinsic Fluorescence.

    PubMed

    Swain, Jitendriya; Kumar Mishra, Ashok

    2015-09-10

    Capsaicin is an ingredient of a wide variety of red peppers, and it has various pharmacological and biological applications. The present study explores the interaction of capsaicin with dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane by monitoring various photophysical parameters using its intrinsic fluorescence. In order to have a clearer understanding of the photophysical responses of capsaicin, studies involving (i) its solvation behavior in different solvents, (ii) the partition coefficient of capsaicin in different thermotropic phase states of lipid bilayer membrane, and (iii) its location inside lipid bilayer membrane have been carried out. Capsaicin has a reasonably high partition coefficient for DMPC liposome membrane, in both solid gel (2.8 ± 0.1 × 10(5)) and liquid crystalline (2.6 ± 0.1 × 10(5)) phases. Fluorescence quenching study using cetylpyridinium chloride (CPC) as quencher suggests that the phenolic group of capsaicin molecule is generally present near the headgroup region and hydrophobic tail present inside hydrophobic core region of the lipid bilayer membrane. The intrinsic fluorescence intensity and lifetime of capsaicin sensitively respond to the temperature dependent phase changes of liposome membrane. Above 15 mol %, capsaicin in the aqueous liposome suspension medium lowers the thermotropic phase transition temperature by about 3 °C, and above 30 mol %, the integrity of the membrane is significantly lost.

  17. Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

    SciTech Connect

    Pradhan, D.; Schlegel, R.A. ); Williamson, P. )

    1991-08-06

    Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho({sup 14}C) ethanolamine (({sup 14}C)AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by ({sup 14}C)AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.

  18. Solute effects on the colloidal and phase behavior of lipid bilayer membranes: ethanol-dipalmitoylphosphatidylcholine mixtures.

    PubMed Central

    Vierl, U; Löbbecke, L; Nagel, N; Cevc, G

    1994-01-01

    By means of the scanning differential calorimetry, x-ray diffractometry, and the dynamic light scattering, we have systematically studied the phase and packing properties of dipalmitoylphosphatidylcholine vesicles or multibilayers in the presence of ethanol. We have also determined the partial ternary phase diagram of such dipalmitoylphosphatidylcholine/water/ethanol mixtures. The directly measured variability of the structural bilayer parameters implies that ethanol binding to the phospholipid bilayers increases the lateral as well as the transverse repulsion between the lipid molecules. This enlarges the hydrocarbon tilt (by up to 23 degrees) and molecular area (by < or = 40%). Ethanol-phospholid association also broadens the interface and, thus, promotes lipid headgroup solvation. This results in excessive swelling (by 130%) of the phosphatidylcholine bilayers in aqueous ethanol solutions. Lateral bilayer expansion, moreover, provokes a successive interdigitation of the hydrocarbon chains in the systems with bulk ethanol concentrations of 0.4-1.2 M. The hydrocarbon packing density as well as the propensity for the formation of lamellar gel phases simultaneously increase. The pretransition temperature of phosphatidylcholine bilayers is more sensitive to the addition of alcohol (initial shift: delta Tp = 22 degrees C/mol) than the subtransition temperature (delta Ts reversible 5 degrees C/mol), whereas the chain-melting phase transition temperature is even less affected (delta Tm = 1.8 degrees C/mol). After an initial decrease of 3 degrees for the bulk ethanol concentrations below 1.2 M, the Tm value increases by 2.5 degrees above this limiting concentration. The gel-phase phosphatidylcholine membranes below Tm are fully interdigitated above this limiting concentration. The chain tilt on the fringe of full chain interdigitation is zero and increases with higher ethanol concentrations. Above Tm, some of the lipid molecules are solubilized by the bound ethanol

  19. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

    PubMed Central

    Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  20. Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

    DOE PAGES

    Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; Lee, Sang -Wook; Sohn, Youngjoo; Oh, Sang -Hyun; Parikh, Atul N.; Lee, Sin -Doo

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

  1. Folding of β-barrel membrane proteins in lipid bilayers - Unassisted and assisted folding and insertion.

    PubMed

    Kleinschmidt, Jörg H

    2015-09-01

    In cells, β-barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert. Model systems have been established to investigate the mechanisms of insertion and folding of these versatile proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers. In these experiments, insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with molecular chaperones from cells. This review is aimed to provide an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid-protein interactions and the function of molecular chaperones and folding assistants. This article is part of a Special Issue entitled: Lipid-protein interactions.

  2. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

    PubMed

    Ryu, Yong-Sang; Wittenberg, Nathan J; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  3. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

    PubMed

    Ryu, Yong-Sang; Wittenberg, Nathan J; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N; Lee, Sin-Doo

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.

  4. Kinetics and thermodynamics of chlorpromazine interaction with lipid bilayers: effect of charge and cholesterol.

    PubMed

    Martins, Patrícia T; Velazquez-Campoy, Adrian; Vaz, Winchil L C; Cardoso, Renato M S; Valério, Joana; Moreno, Maria João

    2012-03-01

    Passive transport across cell membranes is the major route for the permeation of xenobiotics through tight endothelia such as the blood–brain barrier. The rate of passive permeation through lipid bilayers for a given drug is therefore a critical step in the prediction of its pharmacodynamics. We describe a detailed study on the kinetics and thermodynamics for the interaction of chlorpromazine (CPZ), an antipsychotic drug used in the treatment of schizophrenia, with neutral and negatively charged lipid bilayers. Isothermal titration calorimetry was used to study the partition and translocation of CPZ in lipid membranes composed of pure POPC, POPC:POPS (9:1), and POPC:Chol:POPS (6:3:1). The membrane charge due to the presence of POPS as well as the additional charge resulting from the introduction of CPZ in the membrane were taken into account, allowing the calculation of the intrinsic partition coefficients (K(P)) and the enthalpy change (ΔH) associated with the process. The enthalpy change upon partition to all lipid bilayers studied is negative, but a significant entropy contribution was also observed for partition to the neutral membrane. Because of the positive charge of CPZ, the presence of negatively charged lipids in the bilayer increases both the observed amount of CPZ that partitions to the membrane (KP(obs)) and the magnitude of ΔH. However, when the electrostatic effects are discounted, the intrinsic partition coefficient was smaller, indicating that the hydrophobic contribution was less significant for the negatively charged membrane. The presence of cholesterol strongly decreases the affinity of CPZ for the bilayer in terms of both the amount of CPZ that associates with the membrane and the interaction enthalpy. A quantitative characterization of the rate of CPZ translocation through membranes composed of pure POPC and POPC:POPS (9:1) was also performed using an innovative methodology developed in this work based on the kinetics of the heat evolved

  5. Bilayer lipid membranes: An experimental system for biomolecular electronic devices development

    NASA Astrophysics Data System (ADS)

    Ottova-Leitmannova, A.; Ti Tien, H.

    1992-12-01

    The lipid bilayer postulated as the basic structural matrix of biological membranes is widely accepted. At present, the planar bilayer lipid membrane (BLM) together with spherical lipid bilayers (liposomes), upon suitable modification, serves as a most appropriate model for biological membranes. In recent years, advances in microelectronics and interest in ultrathin organic films, including BLMs and Langmuir-Blodgett (L-B) films, have resulted in a unique fusion of ideas toward the development of biosensors and transducers. Furthermore, recent trends in interdisciplinary studies in chemistry, electronics, and biology have led to a new field of research: biomolecular electronics. This exciting new field of scientific-technological endeavor is part of a more general approach toward the development of a new, post-semiconductor electronic technology, namely, molecular electronics with a long-term goal of molecular computers. Recently, it has been demonstrated that BLMs, after suitable modification, can function as electrodes and exhibit nonlinear electronic properties. These and other experimental findings relevant to sensor development and to “biomolecular electronic devices” (BED) will be described in more details in the present review article. Also the potential use of the BLM system together with its modifications in the development of a new class of organic diodes, switches, biosensors, electrochemical photocells, and biofuel cells will be discussed. Additionally, this paper reports also a novel technique for obtaining BLMs (or lipid bilayers) on solid supports. The presence of solid support on one side of the BLM greatly enhances its mechanical stability, while retaining the dynamic properties of the lipid bilayer. Advantages of the new techniques for self-assembling amphiphilic molecules on rigid substrates are discussed in terms of their possible uses. It is evident that the new BLM system (s-BLMs) is potentially useful for technological applications in the

  6. Adsorption kinetics dictate monolayer self-assembly for both lipid-in and lipid-out approaches to droplet interface bilayer formation

    SciTech Connect

    Venkatesan, Guru A.; Lee, Joonho; Farimani, Amir Barati; Heiranian, Mohammad; Collier, C. Patrick; Narayana, Aluru; Sarles, Stephen A.

    2015-11-10

    The droplet interface bilayer (DIB) is a method to assemble planar lipid bilayer membranes between lipid-coated aqueous droplets and has gained popularity among researchers in many fields. Well-packed lipid monolayer on aqueous droplet–oil interfaces is a prerequisite for successfully assembling DIBs. Such monolayers can be achieved by two different techniques: “lipid-in”, in which phospholipids in the form of liposomes are placed in water, and “lipid-out”, in which phospholipids are placed in oil as inverse micelles. While both approaches are capable of monolayer assembly needed for bilayer formation, droplet pairs assembled with these two techniques require significantly different incubation periods and exhibit different success rates for bilayer formation. In our study, we combine experimental interfacial tension measurements with molecular dynamics simulations of phospholipids (DPhPC and DOPC) assembled from water and oil origins to understand the differences in kinetics of monolayer formation. With the results from simulations and by using a simplified model to analyze dynamic interfacial tensions, we conclude that, at high lipid concentrations common to DIBs, monolayer formation is simple adsorption controlled for lipid-in technique, whereas it is predominantly adsorption-barrier controlled for the lipid-out technique due to the interaction of interface-bound lipids with lipid structures in the subsurface. The adsorption barrier established in lipid-out technique leads to a prolonged incubation time and lower bilayer formation success rate, proving a good correlation between interfacial tension measurements and bilayer formation. Also, we clarify that advective flow expedites monolayer formation and improves bilayer formation success rate by disrupting lipid structures, rather than enhancing diffusion, in the subsurface and at the interface for lipid-out technique. Additionally, electrical properties of DIBs formed with varying lipid placement and

  7. Adsorption kinetics dictate monolayer self-assembly for both lipid-in and lipid-out approaches to droplet interface bilayer formation

    DOE PAGES

    Venkatesan, Guru A.; Lee, Joonho; Farimani, Amir Barati; Heiranian, Mohammad; Collier, C. Patrick; Narayana, Aluru; Sarles, Stephen A.

    2015-11-10

    The droplet interface bilayer (DIB) is a method to assemble planar lipid bilayer membranes between lipid-coated aqueous droplets and has gained popularity among researchers in many fields. Well-packed lipid monolayer on aqueous droplet–oil interfaces is a prerequisite for successfully assembling DIBs. Such monolayers can be achieved by two different techniques: “lipid-in”, in which phospholipids in the form of liposomes are placed in water, and “lipid-out”, in which phospholipids are placed in oil as inverse micelles. While both approaches are capable of monolayer assembly needed for bilayer formation, droplet pairs assembled with these two techniques require significantly different incubation periods andmore » exhibit different success rates for bilayer formation. In our study, we combine experimental interfacial tension measurements with molecular dynamics simulations of phospholipids (DPhPC and DOPC) assembled from water and oil origins to understand the differences in kinetics of monolayer formation. With the results from simulations and by using a simplified model to analyze dynamic interfacial tensions, we conclude that, at high lipid concentrations common to DIBs, monolayer formation is simple adsorption controlled for lipid-in technique, whereas it is predominantly adsorption-barrier controlled for the lipid-out technique due to the interaction of interface-bound lipids with lipid structures in the subsurface. The adsorption barrier established in lipid-out technique leads to a prolonged incubation time and lower bilayer formation success rate, proving a good correlation between interfacial tension measurements and bilayer formation. Also, we clarify that advective flow expedites monolayer formation and improves bilayer formation success rate by disrupting lipid structures, rather than enhancing diffusion, in the subsurface and at the interface for lipid-out technique. Additionally, electrical properties of DIBs formed with varying lipid

  8. Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis.

    PubMed

    Funakoshi, Kei; Suzuki, Hiroaki; Takeuchi, Shoji

    2006-12-15

    Artificial planar lipid bilayers are a powerful tool for the functional study of membrane proteins, yet they have not been widely used due to their low stability and reproducibility. This paper describes an accessible method to form a planar lipid bilayer, simply by contacting two monolayers assembled at the interface between water and organic solvent in a microfluidic chip. The membrane of an organic solvent containing phospholipids at the interface was confirmed to be a bilayer by the capacitance measurement and by measuring the ion channel signal from reconstituted antibiotic peptides. We present two different designs for bilayer formation. One equips two circular wells connected, in which the water/solvent/water interface was formed by simply injecting a water droplet into each well. Another equips the cross-shaped microfluidic channel. In the latter design, formation of the interface at the sectional area was controlled by external syringe pumps. Both methods are extremely simple and reproducible, especially in microdevices, and will lead to automation and multiple bilayer formation for the high-throughput screening of membrane transport in physiological and pharmaceutical studies.

  9. Hydrophobic matching between melittin and phosphocholine lipid bilayers having different thicknesses

    NASA Astrophysics Data System (ADS)

    Heller, William; Qian, Shuo

    2014-03-01

    The lipid bilayer of the cellular membrane is more than a simple medium that houses proteins with specific function. Instead, it is an elastic medium that plays an active role in the function of the membrane and that both drives the function of membrane proteins and alters its properties in response to their presence. The conceptual simplicity of membrane active peptides makes them attractive model systems for studying membrane-protein interactions. Melittin, a 27 amino acid cationic peptide having a helix-hinge-helix motif, is one of the most extensively studied examples. Small-angle neutron scattering (SANS) measurements of melittin associated with lipid bilayer vesicles having different hydrocarbon thicknesses showed that the bilayer thickness stretches to match the thickness of the peptide in a manner consistent with a rigid, extended melittin having its helical axis oriented parallel to the bilayer normal. This behavior is surprising considering the helix-hinge-helix motif of the peptide and in contrast to studies indicating that transmembrane helices tilt with respect to the bilayer normal to accommodate differences in hydrophobic thicknesses. Possible sources of the discrepancy will be discussed and explored. This research at SNS and HFIR of ORNL was supported by the US Department of Energy Office of Basic Energy Sciences and the Office of Biological and Environmental Research (ERKP291).

  10. The Water Permeability and Pore Entrance Structure of Aquaporin-4 Depend on Lipid Bilayer Thickness.

    PubMed

    Tong, Jihong; Wu, Zhe; Briggs, Margaret M; Schulten, Klaus; McIntosh, Thomas J

    2016-07-12

    Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10(-13) cm(3)/s (mean ± SE), 3.0 ± 0.3 × 10(-13) cm(3)/s, 2.5 ± 0.2 × 10(-13) cm(3)/s, and 0.9 ± 0.1 × 10(-13) cm(3)/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10(-13) cm(3)/s and 2.5 ± 0.1 × 10(-13) cm(3)/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and α-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon

  11. Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip

    PubMed Central

    Saha, Shimul C.; Powl, Andrew M.; Wallace, B. A.; de Planque, Maurits R. R.; Morgan, Hywel

    2015-01-01

    We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiological screening of ion channels and transporters. A passive pumping method is used to flow microliter volumes of ligand solution across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 μl/min. Phospholipid bilayers are formed across a photolithographically defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with β-cyclodextrin (β-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for determining IC50 values. The assays are fast (30 min for a complete IC50 curve) and simple and require very small amounts of compounds (100 μg in 15 μl). The technology can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores. PMID:25610515

  12. Modeling Yeast Organelle Membranes and How Lipid Diversity Influences Bilayer Properties.

    PubMed

    Monje-Galvan, Viviana; Klauda, Jeffery B

    2015-11-17

    Membrane lipids are important for the health and proper function of cell membranes. We have improved computational membrane models for specific organelles in yeast Saccharomyces cerevisiae to study the effect of lipid diversity on membrane structure and dynamics. Previous molecular dynamics simulations were performed by Jo et al. [(2009) Biophys J. 97, 50-58] on yeast membrane models having six lipid types with compositions averaged between the endoplasmic reticulum (ER) and the plasma membrane (PM). We incorporated ergosterol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol lipids in our models to better describe the unique composition of the PM, ER, and trans-Golgi network (TGN) bilayers of yeast. Our results describe membrane structure based on order parameters (SCD), electron density profiles (EDPs), and lipid packing. The average surface area per lipid decreased from 63.8 ± 0.4 Å(2) in the ER to 47.1 ± 0.3 Å(2) in the PM, while the compressibility modulus (KA) varied in the opposite direction. The high SCD values for the PM lipids indicated a more ordered bilayer core, while the corresponding lipids in the ER and TGN models had lower parameters by a factor of at least 0.7. The hydrophobic core thickness (2DC) as estimated from EDPs is the thickest for PM, which is in agreement with estimates of hydrophobic regions of transmembrane proteins from the Orientation of Proteins in Membranes database. Our results show the importance of lipid diversity and composition on a bilayer's structural and mechanical properties, which in turn influences interactions with the proteins and membrane-bound molecules.

  13. Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

    PubMed

    Rebaud, Samuel; Maniti, Ofelia; Girard-Egrot, Agnès P

    2014-12-01

    Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes.

  14. Charge Equilibration Force Fields for Molecular Dynamics Simulations of Lipids, Bilayers, and Integral Membrane Protein Systems

    PubMed Central

    Lucas, Timothy R.; Bauer, Brad A.; Patel, Sandeep

    2014-01-01

    With the continuing advances in computational hardware and novel force fields constructed using quantum mechanics, the outlook for non-additive force fields is promising. Our work in the past several years has demonstrated the utility of polarizable force fields, those based on the charge equilibration formalism, for a broad range of physical and biophysical systems. We have constructed and applied polarizable force fields for lipids and lipid bilayers. In this review of our recent work, we discuss the formalism we have adopted for implementing the charge equilibration (CHEQ) method for lipid molecules. We discuss the methodology, related issues, and briefly discuss results from recent applications of such force fields. Application areas include DPPC-water monolayers, potassium ion permeation free energetics in the gramicidin A bacterial channel, and free energetics of permeation of charged amino acid analogues across the water-bilayer interface. PMID:21967961

  15. Charge equilibration force fields for molecular dynamics simulations of lipids, bilayers, and integral membrane protein systems.

    PubMed

    Lucas, Timothy R; Bauer, Brad A; Patel, Sandeep

    2012-02-01

    With the continuing advances in computational hardware and novel force fields constructed using quantum mechanics, the outlook for non-additive force fields is promising. Our work in the past several years has demonstrated the utility of polarizable force fields, those based on the charge equilibration formalism, for a broad range of physical and biophysical systems. We have constructed and applied polarizable force fields for lipids and lipid bilayers. In this review of our recent work, we discuss the formalism we have adopted for implementing the charge equilibration (CHEQ) method for lipid molecules. We discuss the methodology, related issues, and briefly discuss results from recent applications of such force fields. Application areas include DPPC-water monolayers, potassium ion permeation free energetics in the gramicidin A bacterial channel, and free energetics of permeation of charged amino acid analogs across the water-bilayer interface. This article is part of a Special Issue entitled: Membrane protein structure and function.

  16. Nitrous oxide vibrational energy relaxation is a probe of interfacial water in lipid bilayers.

    PubMed

    Chieffo, Logan R; Shattuck, Jeffrey T; Pinnick, Eric; Amsden, Jason J; Hong, M K; Wang, Feng; Erramilli, Shyamsunder; Ziegler, Lawrence D

    2008-10-01

    Ultrafast infrared spectroscopy of N 2O is shown to be a sensitive probe of hydrophobic and aqueous sites in lipid bilayers. Distinct rates of VER of the nu 3 antisymmetric stretching mode of N 2O can be distinguished for N 2O solvated in the acyl tail, interfacial water, and bulk water regions of hydrated dioleoylphosphatidylcholine (DOPC) bilayers. The lifetime of the interfacial N 2O population is hydration-dependent. This effect is attributed to changes in the density of intermolecular states resonant with the nu 3 band ( approximately 2230 cm (-1)) resulting from oriented interfacial water molecules near the lipid phosphate. Thus, the N 2O VER rate becomes a novel and experimentally convenient tool for reporting on the structure and dynamics of interfacial water in lipids and, potentially, in other biological systems. PMID:18793010

  17. Self-assembly formation of lipid bilayer coatings on bare aluminum oxide: overcoming the force of interfacial water.

    PubMed

    Jackman, Joshua A; Tabaei, Seyed R; Zhao, Zhilei; Yorulmaz, Saziye; Cho, Nam-Joon

    2015-01-14

    Widely used in catalysis and biosensing applications, aluminum oxide has become popular for surface functionalization with biological macromolecules, including lipid bilayer coatings. However, it is difficult to form supported lipid bilayers on aluminum oxide, and current methods require covalent surface modification, which masks the interfacial properties of aluminum oxide, and/or complex fabrication techniques with specific conditions. Herein, we addressed this issue by identifying simple and robust strategies to form fluidic lipid bilayers on aluminum oxide. The fabrication of a single lipid bilayer coating was achieved by two methods, vesicle fusion under acidic conditions and solvent-assisted lipid bilayer (SALB) formation under near-physiological pH conditions. Importantly, quartz crystal microbalance with dissipation (QCM-D) monitoring measurements determined that the hydration layer of a supported lipid bilayer on aluminum oxide is appreciably thicker than that of a bilayer on silicon oxide. Fluorescence recovery after photobleaching (FRAP) analysis indicated that the diffusion coefficient of lateral lipid mobility was up to 3-fold greater on silicon oxide than on aluminum oxide. In spite of this hydrodynamic coupling, the diffusion coefficient on aluminum oxide, but not silicon oxide, was sensitive to the ionic strength condition. Extended-DLVO model calculations estimated the thermodynamics of lipid-substrate interactions on aluminum oxide and silicon oxide, and predict that the range of the repulsive hydration force is greater on aluminum oxide, which in turn leads to an increased equilibrium separation distance. Hence, while a strong hydration force likely contributes to the difficulty of bilayer fabrication on aluminum oxide, it also confers advantages by stabilizing lipid bilayers with thicker hydration layers due to confined interfacial water. Such knowledge provides the basis for improved surface functionalization strategies on aluminum oxide

  18. Self-assembly formation of lipid bilayer coatings on bare aluminum oxide: overcoming the force of interfacial water.

    PubMed

    Jackman, Joshua A; Tabaei, Seyed R; Zhao, Zhilei; Yorulmaz, Saziye; Cho, Nam-Joon

    2015-01-14

    Widely used in catalysis and biosensing applications, aluminum oxide has become popular for surface functionalization with biological macromolecules, including lipid bilayer coatings. However, it is difficult to form supported lipid bilayers on aluminum oxide, and current methods require covalent surface modification, which masks the interfacial properties of aluminum oxide, and/or complex fabrication techniques with specific conditions. Herein, we addressed this issue by identifying simple and robust strategies to form fluidic lipid bilayers on aluminum oxide. The fabrication of a single lipid bilayer coating was achieved by two methods, vesicle fusion under acidic conditions and solvent-assisted lipid bilayer (SALB) formation under near-physiological pH conditions. Importantly, quartz crystal microbalance with dissipation (QCM-D) monitoring measurements determined that the hydration layer of a supported lipid bilayer on aluminum oxide is appreciably thicker than that of a bilayer on silicon oxide. Fluorescence recovery after photobleaching (FRAP) analysis indicated that the diffusion coefficient of lateral lipid mobility was up to 3-fold greater on silicon oxide than on aluminum oxide. In spite of this hydrodynamic coupling, the diffusion coefficient on aluminum oxide, but not silicon oxide, was sensitive to the ionic strength condition. Extended-DLVO model calculations estimated the thermodynamics of lipid-substrate interactions on aluminum oxide and silicon oxide, and predict that the range of the repulsive hydration force is greater on aluminum oxide, which in turn leads to an increased equilibrium separation distance. Hence, while a strong hydration force likely contributes to the difficulty of bilayer fabrication on aluminum oxide, it also confers advantages by stabilizing lipid bilayers with thicker hydration layers due to confined interfacial water. Such knowledge provides the basis for improved surface functionalization strategies on aluminum oxide

  19. Dependence of norfloxacin diffusion across bilayers on lipid composition.

    PubMed

    Purushothaman, Sowmya; Cama, Jehangir; Keyser, Ulrich F

    2016-02-21

    Antibiotic resistance is a growing concern in medicine and raises the need to develop and design new drug molecules that can efficiently inhibit bacterial replication. Spurring the passive uptake of the drug molecules is an obvious solution. However our limited understanding of drug-membrane interactions due to the presence of an overwhelming variety of lipids constituting cellular membranes and the lack of facile tools to probe the bio-physical interactions between drugs and lipids imposes a major challenge towards developing new drug molecules that can enter the cell via passive diffusion. Here, we used a label-free micro-fluidic platform combined with giant unilamellar lipid vesicles to investigate the permeability of membranes containing mixtures of DOPE and DOPG in DOPC, leading to a label-free measurement of passive membrane-permeability of autofluorescent antibiotics. A fluoroquinolone drug, norfloxacin was used as a case study. Our results indicate that the diffusion of norfloxacin is strongly dependent on the lipid composition which is not expected from the traditional octanol-lipid partition co-efficient assay. The anionic lipid, DOPG, slows the diffusion process whereas the diffusion across liposomes containing DOPE increases with higher DOPE concentration. Our findings emphasise the need to investigate drug-membrane interactions with focus on the specificity of drugs to lipids for efficient drug delivery, drug encapsulation and targeted drug-delivery.

  20. Ion channels induced in lipid bilayers by subvirion particles of the nonenveloped mammalian reoviruses.

    PubMed Central

    Tosteson, M T; Nibert, M L; Fields, B N

    1993-01-01

    Mechanisms by which nonenveloped viruses penetrate cell membranes as an early step in infection are not well understood. Current ideas about the mode for cytosolic penetration by nonenveloped viruses include (i) formation of a membrane-spanning pore through which viral components enter the cell and (ii) local breakdown of the cellular membrane to provide direct access of infecting virus to the cell's interior. Here we report that of the three viral particles of nonenveloped mammalian reoviruses: virions, infectious subvirion particles, and cores (the last two forms generated from intact reovirus virions by proteolysis), only the infectious subvirion particles induced the formation of anion-selective, multisized channels in planar lipid bilayers under the experimental conditions used in this study. The value for the smallest size conductance varied depending on the lipid composition of the bilayer between 90 pS (Asolectin) and 300 pS (phosphatidylethanolamine:phosphatidylserine) and was found to be voltage independent. These findings are consistent with a proposal that the proteolytically activated infectious subviral particles mediate the interaction between virus and the lipid bilayer of a cell membrane during penetration. In addition, the findings indicate that the "penetration proteins" of some enveloped and nonenveloped viruses share similarities in the way they interact with bilayers. PMID:7504268

  1. Direct observation and control of supported lipid bilayer formation with interferometric scattering microscopy.

    PubMed

    Andrecka, Joanna; Spillane, Katelyn M; Ortega-Arroyo, Jaime; Kukura, Philipp

    2013-12-23

    Supported lipid bilayers (SLB) are frequently used to study processes associated with or mediated by lipid membranes. The mechanism by which SLBs form is a matter of debate, largely due to the experimental difficulty associated with observing the adsorption and rupture of individual vesicles. Here, we used interferometric scattering microscopy (iSCAT) to directly visualize membrane formation from nanoscopic vesicles in real time. We observed a number of previously proposed phenomena such as vesicle adsorption, rupture, movement, and a wave-like bilayer spreading. By varying the vesicle size and the lipid-surface interaction strength, we rationalized and tuned the relative contributions of these phenomena to bilayer formation. Our results support a model where the interplay between bilayer edge tension and the overall interaction energy with the surface determine the mechanism of SLB formation. The unique combination of sensitivity, speed, and label-free imaging capability of iSCAT provides exciting prospects not only for investigations of SLB formation, but also for studies of assembly and disassembly processes on the nanoscale with previously unattainable accuracy and sensitivity.

  2. Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

    PubMed Central

    Chen, Yuhui; Huang, Shengjian; Liu, Dongfang

    2015-01-01

    The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution. PMID:25741636

  3. Holographic interferometry of ultrasmall-pressure-induced curvature changes of bilayer lipid membranes

    SciTech Connect

    Picard, G.; Schneider-Henriquez, J.E.; Fendler, J.H. )

    1990-01-25

    Two-exposure interferometric holograms have been shown to sensitively report ultrasmall-pressure (10 natm)-induced curvature changes in glyceryl monooleate (GMO) bilayer lipid membranes (BLMs). The number of concentric fringes observed, and hence the lateral distance between the plane of the Teflon and the BLM, increased linearly with increasing transmembrane pressure and led to a value of 1.1 {plus minus} 0.05 dyn/cm for the surface tension of the BLM. BLMs with appreciable Plateau-Gibbs borders have been shown to undergo nonuniform deformation; the bilayer portion is distorted less than the surrounding Plateau-Gibbs border upon the application of a transmembrane pressure gradient.

  4. Size dependence of gold nanoparticle interactions with a supported lipid bilayer: A QCM-D study.

    PubMed

    Bailey, Christina M; Kamaloo, Elaheh; Waterman, Kellie L; Wang, Kathleen F; Nagarajan, Ramanathan; Camesano, Terri A

    2015-01-01

    Knowledge of nanoparticle (NP)-membrane interactions is important to advances in nanomedicine as well as for determining the safety of NPs to humans and the ecosystem. This study focuses on a unique mechanism of cytotoxicity, cell membrane destabilization, which is principally dependent on the nanoparticle nature of the material rather than on its molecular properties. We investigated the interactions of 2, 5, 10, and 40nm gold NPs with supported lipid bilayer (SLB) of L-α-phosphatidylcholine using quartz crystal microbalance with dissipation monitoring (QCM-D). Gold NPs were tested both in the absence of and in the presence of polymethacrylic acid (PMAA), used to simulate the natural organic matter (NOM) in the environment. In the absence of PMAA, for all NP sizes, we observed only small mass losses (1 to 6ng) from the membrane. This small lipid removal may be a free energy lowering mechanism to relieve stresses induced by the adsorption of NPs, with the changes too small to affect the membrane integrity. In the presence of PMAA, we observed a net mass increase in the case of smaller NPs. We suggest that the increased adhesion between the NP and the bilayer, promoted by PMAA, causes sufficient NP adsorption on the bilayer to overcompensate for any loss of lipid. The most remarkable observation is the significant mass loss (60ng) for the case of 40nm NPs. We attribute this to the lipid bilayer engulfing the NP and leaving the crystal surface. We propose a simple phenomenological model to describe the competition between the particle-bilayer adhesion energy, the bilayer bending energy, and the interfacial energy at bilayer defect edges. The model shows that the larger NPs, which become more adhesive because of the polymer adsorption, are engulfed by the bilayer and leave the crystal surface, causing large mass loss and membrane disruption. The QCM-D measurements thus offer direct evidence that even if NPs are intrinsically not cytotoxic, they can become cytotoxic

  5. Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

    PubMed Central

    Tabaei, Seyed R.; Jackman, Joshua A.; Kim, Minchul; Yorulmaz, Saziye; Vafaei, Setareh; Cho, Nam-Joon

    2015-01-01

    In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting

  6. Electron density analysis of the effects of sugars on the structure of lipid bilayers at low hydration - a preliminary study

    SciTech Connect

    Lenné, T.; Kent, B.; Koster, K.L.; Garvey, C.J.; Bryant, G.

    2012-02-06

    Small angle X-ray scattering is used to study the effects of sugars on membranes during dehydration. Previous work has shown that the bilayer and chain-chain repeat spacings of DPPC bilayers are relatively unaffected by the presence of sugars. In this work we present a preliminary analysis of the electron density profiles of DPPC in the presence of sugars at low hydration. The difficulties of determining the correct phasing are discussed. Sugars and other small solutes have been shown to have an important role in improving the tolerance of a range of species to desiccation and freezing. In particular it has been shown that sugars can stabilize membranes in the fluid membrane phase during dehydration, and in the fully dehydrated state. Equivalently, at a particular hydration, the presence of sugars lowers the transition temperature between the fluid and gel phases. There are two competing models for explaining the effects of sugars on membrane phase transition temperatures. One, designated the water replacement hypothesis (WRH) states that sugars hydrogen bond to phospholipid headgroups, thus hindering the fluid-gel phase transition. One version of this model suggests that certain sugars (such as trehalose) achieve the measured effects by inserting between the phospholipid head groups. An alternative model explains the observed effects of sugars in terms of the sugars effect on the hydration repulsion that develops between opposing membranes during dehydration. The hydration repulsion leads to a lateral compressive stress in the bilayer which squeezes adjacent lipids more closely together, resulting in a transition to the gel phase. When sugars are present, their osmotic and volumetric effects reduce the hydration repulsion, reduce the compressive stress in the membranes, and therefore tend to maintain the average lateral separation between lipids. This model is called the hydration forces explanation (HFE). We recently showed that neither mono- nor di

  7. Self-Spreading of Lipid Bilayer on a Hydrophobic Surface Made by Self-Assembled Monolayer with Short Alkyl Chain.

    PubMed

    Omori, Yuya; Sakaue, Hiroyuki; Takahagi, Takayuki; Suzuki, Hitoshi

    2016-04-01

    Behaviors of self-spreading of lipid bilayer membrane on a glass surface modified with self-assembled monolayer (SAM) with short alkyl chain were observed with fluorescence microscopy. Hydrophobic surface made by SAM was found to hamper the self-spreading phenomenon but the lipid bilayer spread on a hydrophilic one where SAM was decomposed by oxidation. On a binary surface having a hydrophobic region and a hydrophilic one, the lipid bilayer spread on the hydrophilic region but it stopped at the boundary of the hydrophobic region.

  8. Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation.

    PubMed

    Jalili, Seifollah; Saeedi, Marzieh

    2016-01-01

    Liposomal formulation of curcumin is an important therapeutic agent for the treatment of various cancers. Despite extensive studies on the biological effects of this formulation in cancer treatment, much remains unknown about curcumin-liposome interactions. Understanding how different lipid bilayers respond to curcumin molecule may help us to design more effective liposomal curcumin. Here, we used molecular dynamics simulation method to investigate the behavior of curcumin in two lipid bilayers commonly used in preparation of liposomal curcumin, namely dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG). First, the free energy barriers for translocation of one curcumin molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). The computed free energy profile exhibits a global minimum at the solvent-headgroup interface (LH region) for both lipid membranes. We also evaluated the free energy difference between the equilibrium position of curcumin in the lipid bilayer and bulk water as the excess chemical potential. Our results show that curcumin has the higher affinity in DMPG compared to DPPC lipid bilayer (-8.39 vs. -1.69 kBT) and this is related to more hydrogen bond possibility for curcumin in DMPG lipid membrane. Next, using an unconstrained molecular dynamic simulation with curcumin initially positioned at the center of lipid bilayer, we studied various properties of each lipid bilayer system in the presence of curcumin molecule that was in full agreement with PMF and experimental data. The results of these simulation studies suggest that membrane composition could have a large effect on interaction of curcumin-lipid bilayer.

  9. Linking lipid architecture to bilayer structure and mechanics using self-consistent field modelling

    SciTech Connect

    Pera, H.; Kleijn, J. M.; Leermakers, F. A. M.

    2014-02-14

    To understand how lipid architecture determines the lipid bilayer structure and its mechanics, we implement a molecularly detailed model that uses the self-consistent field theory. This numerical model accurately predicts parameters such as Helfrichs mean and Gaussian bending modulus k{sub c} and k{sup ¯} and the preferred monolayer curvature J{sub 0}{sup m}, and also delivers structural membrane properties like the core thickness, and head group position and orientation. We studied how these mechanical parameters vary with system variations, such as lipid tail length, membrane composition, and those parameters that control the lipid tail and head group solvent quality. For the membrane composition, negatively charged phosphatidylglycerol (PG) or zwitterionic, phosphatidylcholine (PC), and -ethanolamine (PE) lipids were used. In line with experimental findings, we find that the values of k{sub c} and the area compression modulus k{sub A} are always positive. They respond similarly to parameters that affect the core thickness, but differently to parameters that affect the head group properties. We found that the trends for k{sup ¯} and J{sub 0}{sup m} can be rationalised by the concept of Israelachivili's surfactant packing parameter, and that both k{sup ¯} and J{sub 0}{sup m} change sign with relevant parameter changes. Although typically k{sup ¯}<0, membranes can form stable cubic phases when the Gaussian bending modulus becomes positive, which occurs with membranes composed of PC lipids with long tails. Similarly, negative monolayer curvatures appear when a small head group such as PE is combined with long lipid tails, which hints towards the stability of inverse hexagonal phases at the cost of the bilayer topology. To prevent the destabilisation of bilayers, PG lipids can be mixed into these PC or PE lipid membranes. Progressive loading of bilayers with PG lipids lead to highly charged membranes, resulting in J{sub 0}{sup m}≫0, especially at low ionic

  10. Effects of Sugars on Lipid Bilayers during Dehydration − SAXS/WAXS Measurements and Quantitative Model

    SciTech Connect

    Lenne, Thomas; Garvey, Christopher J.; Koster, Karen L.; Bryant, Gary

    2009-04-02

    We present an X-ray scattering study of the effects of dehydration on the bilayer and chain-chain repeat spacings of dipalmitoylphosphatidylcholine bilayers in the presence of sugars. The presence of sugars has no effect on the average spacing between the phospholipid chains in either the fluid or gel phase. Using this finding, we establish that for low sugar concentrations only a small amount of sugar exclusion occurs. Under these conditions, the effects of sugars on the membrane transition temperatures can be explained quantitatively by the reduction in hydration repulsion between bilayers due to the presence of the sugars. Specific bonding of sugars to lipid headgroups is not required to explain this effect.

  11. Experimental verification of lipid bilayer structure through multi-scale modeling.

    PubMed

    Perlmutter, Jason D; Sachs, Jonathan N

    2009-10-01

    Great progress has been made in applying coarse-grain molecular dynamics (CGMD) simulations to the investigation of membrane biophysics. In order to validate the accuracy of CGMD simulations of membranes, atomistic scale detail is necessary for direct comparison to structural experiments. Here, we present our strategy for verifying CGMD lipid bilayer simulations. Through reverse coarse graining and subsequent calculation of the bilayer electron density profile, we are able to compare the simulations to our experimental low angle X-ray scattering (LAXS) data. In order to determine the best match to the experimental data, atomistic simulations are run at a range of areas (in the NP(N)AT ensemble), starting from distinct configurations extracted from the CGMD simulation (run in the NPT ensemble). We demonstrate the effectiveness of this procedure with two small, single-component bilayers, and suggest that the greater utility of our algorithm will be for CGMD simulations of more complex structures. PMID:19616507

  12. Nanoscale mechanical probing of supported lipid bilayers with atomic force microscopy.

    PubMed

    Das, Chinmay; Sheikh, Khizar H; Olmsted, Peter D; Connell, Simon D

    2010-10-01

    We present theory and experiments for the force-distance curve F(z(0)) of an atomic force microscope (AFM) tip (radius R) indenting a supported fluid bilayer (thickness 2d). For realistic conditions the force is dominated by the area compressibility modulus κ(A) of the bilayer and, to an excellent approximation, given by F=πκ(A)Rz(0)(2)/(2d-z(0))(2). The experimental AFM force curves from coexisting liquid ordered and liquid disordered domains in three-component lipid bilayers are well described by our model, which provides κ(A) in agreement with literature values. The liquid ordered phase has a yieldlike response that we model as due to the breaking of hydrogen bonds.

  13. Distribution and dynamics of quinones in the lipid bilayer mimicking the inner membrane of mitochondria.

    PubMed

    Kaurola, Petri; Sharma, Vivek; Vonk, Amanda; Vattulainen, Ilpo; Róg, Tomasz

    2016-09-01

    Quinone and its analogues (Q) constitute an important class of compounds that perform key electron transfer reactions in oxidative- and photo-phosphorylation. In the inner membrane of mitochondria, ubiquinone molecules undergo continuous redox transitions enabling electron transfer between the respiratory complexes. In such a dynamic system undergoing continuous turnover for ATP synthesis, an uninterrupted supply of substrate molecules is absolutely necessary. In the current work, we have performed atomistic molecular dynamics simulations and free energy calculations to assess the structure, dynamics, and localization of quinone and its analogues in a lipid bilayer, whose composition mimics the one in the inner mitochondrial membrane. The results show that there is a strong tendency of both quinone and quinol molecules to localize in the vicinity of the lipids' acyl groups, right under the lipid head group region. Additionally, we observe a second location in the middle of the bilayer where quinone molecules tend to stabilize. Translocation of quinone through a lipid bilayer is very fast and occurs in 10-100ns time scale, whereas the translocation of quinol is at least an order of magnitude slower. We suggest that this has important mechanistic implications given that the localization of Q ensures maximal occupancy of the Q-binding sites or Q-entry points in electron transport chain complexes, thereby maintaining an optimal turnover rate for ATP synthesis. PMID:27342376

  14. Directly mapping the surface charge density of lipid bilayers under physiological conditions

    NASA Astrophysics Data System (ADS)

    Fuhs, Thomas; Klausen, Lasse Hyldgaard; Besenbacher, Flemming; Dong, Mingdong

    2015-03-01

    The surface charge density of lipid bilayers governs the cellular uptake of charged particles and guides cell-cell and cell-surface interactions. Direct probing of the potential requires sub nanometer distances as the electrostatic potential is screened by high physiological salt concentrations. This prevented direct measurement of the SCD under physiological conditions. In this study we investigate supported bilayers of lipid mixtures that form domains of distinct surface charges, submerged in 150mM NaCl. We use a scanning ion-conductance microscope (SICM) setup to measure the ionic current through a nanopipette as the pipette is scanned several nanometers above the sample. The charged headgroups of the lipids attract counter ions leading to a charge dependent enhancement of the ion concentration near the surface. This creates a measurable change of conductivity in the vicinity of the surface. As the dependency of the current on the SCD and pipette potential is non-trivial we characterized it using numerical solutions to Poisson and Nernst-Planck equations. Based on the simulation results we propose an imaging method. We confirm feasibility of the proposed method by experimentally mapping the local surface charge density of phase separated lipid bilayers.

  15. Dynamic implicit-solvent coarse-grained models of lipid bilayer membranes: fluctuating hydrodynamics thermostat.

    PubMed

    Wang, Yaohong; Sigurdsson, Jon Karl; Brandt, Erik; Atzberger, Paul J

    2013-08-01

    We introduce a thermostat based on fluctuating hydrodynamics for dynamic simulations of implicit-solvent coarse-grained models of lipid bilayer membranes. We show our fluctuating hydrodynamics approach captures interesting correlations in the dynamics of lipid bilayer membranes that are missing in simulations performed using standard Langevin dynamics. Our momentum conserving thermostat accounts for solvent-mediated momentum transfer by coupling coarse-grained degrees of freedom to stochastic continuum fields that account for both the solvent hydrodynamics and thermal fluctuations. We present both a general framework and specific methods to couple the particle and continuum degrees of freedom in a manner consistent with statistical mechanics and amenable to efficient computational simulation. For self-assembled vesicles, we study the diffusivity of lipids and their spatial correlations. We find the hydrodynamic coupling yields within the bilayer interesting correlations between diffusing lipids that manifest as a vortex-like structure similar to those observed in explicit-solvent simulations. We expect the introduced fluctuating hydrodynamics methods to provide a way to extend implicit-solvent models for use in a wide variety of dynamic studies.

  16. BILAYER LIPID MEMBRANE (BLM) BASED ION SELECTIVE ELECTRODES AT THE MESO, MICRO, AND NANO SCALES

    PubMed Central

    Liu, Bingwen; Rieck, Daniel; Van Wie, Bernard J.; Cheng, Gary J.; Moffett, David F.; Kidwell, David A.

    2009-01-01

    This paper presents a novel method for making micron-sized apertures with tapered sidewalls and nano-sized apertures. Their use in bilayer lipid membrane-based ion selective electrode design is demonstrated and compared to mesoscale bilayers and traditional PVC ion selective electrodes. Micron-sized apertures are fabricated in SU-8 photoresist films and vary in diameter from 10 to 40 microns. The tapered edges in SU-8 films are desired to enhance bilayer lipid membrane (BLM) formation and are fabricated by UV-light overexposure. Nanoapertures are made in boron diffused silicon film. The membranes are used as septa to separate two potassium chloride solutions of different concentrations. Lecithin BLMs are assembled on the apertures by ejecting lipid solution. Potassium ionophore, dibenzo-18-crown-6, is incorporated into BLMs by dissolving it in the lipid solution before membrane assembly. Voltage changes with increasing potassium ion concentrations are recorded with an A/D converter. Various ionophore concentrations in BLMs are investigated. At least a 1% concentration is needed for consistent slopes. Electrode response curves are linear over the 10−6 to 0.1 M range with a sub-Nernstian slope of 20 mV per Log concentration change. This system shows high selectivity to potassium ions over potential interfering sodium ions. BLMs on the three different aperture sizes at the meso, micro, and nano-scales all show similar linear ranges and limits of detection (LODs) as PVC ion selective membranes. PMID:19008091

  17. Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking

    PubMed Central

    Rose, Markus; Hirmiz, Nehad; Moran-Mirabal, Jose M.; Fradin, Cécile

    2015-01-01

    Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 μm2 · s−1 and DSPT ~ 2 μm2 · s−1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D ≳ 1 μm2 · s−1 for FCS and D ≲ 5 μm2 · s−1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes. PMID:26610279

  18. Free-energy determinants of alpha-helix insertion into lipid bilayers.

    PubMed Central

    Ben-Tal, N; Ben-Shaul, A; Nicholls, A; Honig, B

    1996-01-01

    A detailed treatment is provided of the various free-energy terms that contribute to the transfer of a polyalanine alpha-helix from the aqueous phase into lipid bilayers. In agreement with previous work, the hydrophobic effect is found to provide the major driving force for helix insertion. However, an opposing effect of comparable magnitude is also identified and is attributed to the large free-energy penalty associated with the desolvation of peptide hydrogen bonds on transfer to the low dielectric environment of the bilayer. Lipid perturbation effects as well as the entropy loss associated with helix immobilization in the bilayer are also evaluated. Two configurations of a membrane-bound 25mer polyalanine helix were found to be lower in free energy than the isolated helix in the aqueous phase. The first corresponds to the case of vertical insertion, in which a helix terminus protrudes from each side of the bilayer. The second minimum is for the case of horizontal insertion, for which the helix is adsorbed upon the surface of the bilayer. The calculated free-energy minima are found to be in good agreement with recent measurements of related systems. Large free-energy barriers resulting from desolvation of unsatisfied hydrogen-bonding groups at the helix termini are obtained for both insertion processes. The barriers for insertion are significantly reduced if the helix termini are assumed to be "capped" through the formation of hydrogen bonds with polar sidechains. For uncapped helices, our results support recently proposed models in which helices are inserted by first adsorbing on the membrane surface and then having one terminus "swing around" so as to penetrate the bilayer. Images FIGURE 1 PMID:8785340

  19. Membrane-Protein Interactions in a Generic Coarse-Grained Model for Lipid Bilayers

    PubMed Central

    West, Beate; Brown, Frank L.H.; Schmid, Friederike

    2009-01-01

    Abstract We study membrane-protein interactions and membrane-mediated protein-protein interactions by Monte Carlo simulations of a generic coarse-grained model for lipid bilayers with cylindrical hydrophobic inclusions. The strength of the hydrophobic force and the hydrophobic thickness of the proteins are systematically varied. The results are compared with analytical predictions of two popular analytical theories: The Landau-de Gennes theory and the elastic theory. The elastic theory provides an excellent description of the fluctuation spectra of pure membranes and successfully reproduces the deformation profiles of membranes around single proteins. However, its prediction for the potential of mean force between proteins is not compatible with the simulation data for large distances. The simulations show that the lipid-mediated interactions are governed by five competing factors: direct interactions; lipid-induced depletion interactions; lipid bridging; lipid packing; and a smooth long-range contribution. The mechanisms leading to hydrophobic mismatch interactions are critically analyzed. PMID:18835907

  20. Driving force of binding of amyloid {beta}-protein to lipid bilayers

    SciTech Connect

    Ikeda, Keisuke; Matsuzaki, Katsumi

    2008-06-06

    Amyloid {beta}-protein (A{beta}) has been reported to interact with a variety of lipid species, although the thermodynamic driving force remains unclear. We investigated the binding of A{beta}s labeled with the dye diethylaminocoumarin (DAC-A{beta}s) to lipid bilayers under various conditions. DAC-A{beta}-(1-40) electrostatically bound to anionic and cationic lipids at acidic and alkaline interfacial pH, respectively. However, at neutral pH, electroneutral A{beta} did not bind to these lipids, indicating little hydrophobic interaction between A{beta}-(1-40) and the acyl chains of lipids. In contrast, DAC-A{beta} associated with glycolipids even under electroneutral conditions. These results suggested that hydrogen-bonding as well as hydrophobic interactions with sugar groups of glycolipids drive the membrane binding of A{beta}-(1-40)

  1. Acceleration of Lateral Equilibration in Mixed Lipid Bilayers Using Replica Exchange with Solute Tempering

    PubMed Central

    2015-01-01

    The lateral heterogeneity of cellular membranes plays an important role in many biological functions such as signaling and regulating membrane proteins. This heterogeneity can result from preferential interactions between membrane components or interactions with membrane proteins. One major difficulty in molecular dynamics simulations aimed at studying the membrane heterogeneity is that lipids diffuse slowly and collectively in bilayers, and therefore, it is difficult to reach equilibrium in lateral organization in bilayer mixtures. Here, we propose the use of the replica exchange with solute tempering (REST) approach to accelerate lateral relaxation in heterogeneous bilayers. REST is based on the replica exchange method but tempers only the solute, leaving the temperature of the solvent fixed. Since the number of replicas in REST scales approximately only with the degrees of freedom in the solute, REST enables us to enhance the configuration sampling of lipid bilayers with fewer replicas, in comparison with the temperature replica exchange molecular dynamics simulation (T-REMD) where the number of replicas scales with the degrees of freedom of the entire system. We apply the REST method to a cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer mixture and find that the lateral distribution functions of all molecular pair types converge much faster than in the standard MD simulation. The relative diffusion rate between molecules in REST is, on average, an order of magnitude faster than in the standard MD simulation. Although REST was initially proposed to study protein folding and its efficiency in protein folding is still under debate, we find a unique application of REST to accelerate lateral equilibration in mixed lipid membranes and suggest a promising way to probe membrane lateral heterogeneity through molecular dynamics simulation. PMID:25328493

  2. NMR Study of Collective Motions and Bending Rigidity in Multilamellar System of Lipid and Surfactant Bilayers

    NASA Astrophysics Data System (ADS)

    Struppe, J.; Noack, F.; Klose, G.

    1997-10-01

    The frequency dependence of the longitudinal proton spin relaxation time T1 was measured by field-cycling and standard NMR techniques at different temperatures in the liquid crystalline lamellar phases of bilayer systems, composed of lipids, nonionic surfactants, and lipid-surfactant mixtures. We show by our data analysis, comparing various motional models such as layer undulations (LUs) and relaxation by translational diffusion mediated reorientations (TR), that collective layer undulations with their typical T1~ν behaviour determine the low frequency T1 dispersion in both unoriented and glass plate-oriented bilayer systems. The angular dependence of the T1 dispersion for the oriented bilayer system supports these findings and provides a more critical analysis of the two dimensional self-diffusion than in unoriented samples. The evaluated fitting parameters of the LU model allows, together with the measured second moment of the proton NMR signal for the lipid, calculation of the bending rigidity ϰc for these bilayers at different levels of hydration. The obtained values of ϰc turn out to be too large compared with the literature. However, using recent LU models (B. Halle) which include the obvious couplings between neighbouring bilayers at low Larmor frequencies, the corrected ϰc of the fully hydrated membrane systems are comparable to those obtained from the standard videooptical experiments. Therefore proton spin relaxation measure-ments at low Larmor frequencies with the field-cycling technique are a suitable means to determine the bending rigidity ϰc of model membrane systems at low hydrations and of systems containing surfactants.

  3. Dithionite penetration through phospholipid bilayers as a measure of defects in lipid molecular packing.

    PubMed

    Langner, M; Hui, S W

    1993-04-01

    The permeability of dithionite through bilayers was utilized to probe the structural defects in the bilayers of these lipids through their respective gel-fluid and bilayer-hexagonal phase transitions. The water soluble dithionite ion penetrates intact bilayers very slowly. The rate of irreversible quenching of the fluorescence of NBD-PE labelled liposomes may thus be used as an indicator of the permeability of this ion through bilayers. The quenching rate has a fast and a slow component, the fast one corresponds to the quenching of fluorophores immediately accessible to the quencher, i.e. those on the outer surface of liposomes. The slower component represents the average rate of penetration of the quencher through the bilayer, to quench those fluorophores at the inner shells of the multilamellar vesicles. Both rates may be approximated by a single exponential function. The slow exponent is simply related to the permeability. The permeability of DMPC as a function of temperature shows a peak at the gel-fluid phase transition at 24 degrees C, but returns to about the pre-transition value at temperatures above the phase transition. The permeability of egg PE shows a hump at 45 degrees C before the hexagonal phase transition at 65 degrees C is reached and becomes infinite at the hexagonal phase transition as all fluorophores are immediately accessible to the quencher. We believe that the permeability measured by this method relates more to the molecular packing defects which maximizes at the gel-fluid phase transition temperatures just below the bilayer-hexagonal phase transition, rather than the general packing order which simply changes with structural phases.

  4. Interaction of myelin basic protein isoforms with lipid bilayers studied by FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Jackson, Michael; Choo, Lin-P'ing; Boulias, Christopher; Moscarello, Mario A.; Mantsch, Henry H.

    1993-05-01

    The secondary structure of the naturally occurring isoforms of myelin basic protein (MBP1-8) from human myelin was studied by Fourier transform infrared spectroscopy under a variety of experimental conditions. In aqueous solution each isoform was found to be unstructured. In the presence of negatively charged liquid bilayers MBP1-4 were shown to exhibit an amide I band maximum indicative of the adoption of (alpha) -helical secondary structures. A detailed analysis revealed that significant proportions of (beta) -sheet secondary structure were also present. MBP5 and MBP8, which have significantly less cationic charge than MBP1-4, exhibited an amide I maximum identical to that seen in solution, suggesting that no interaction with the bilayer occurred. Analysis of the lipid CH2 and C equals O stretching vibrations also pointed towards significant interaction of MBP1-4 with the bilayer. The changes in intensity and frequency of these bands which typically accompany the phase transition in the pure bilayer were abolished by addition of the proteins. No such effect was seen for MBP5 and 8, the normal lipid phase transition being apparent. The implications of these results in the aetiology of multiple sclerosis is discussed.

  5. Interaction Forces between Supported Lipid Bilayers in the Presence of PEGylated Polymers.

    PubMed

    Banquy, Xavier; Lee, Dong Woog; Kristiansen, Kai; Gebbie, Matthew A; Israelachvili, Jacob N

    2016-01-11

    Using the surface forces apparatus (SFA), interaction forces between supported lipid bilayers were measured in the presence of polyethylene glycol and two other commercially available pegylated triblock polymers, Pluronic F68 and F127. Pluronic F68 has a smaller central hydrophobic block compared to F127 and therefore is more hydrophilic. The study aimed to unravel the effects of polymer architecture and composition on the interactions between the bilayers. Our keys findings show that below the critical aggregation concentration (CAC) of the polymers, a soft, weakly anchored, polymer layer is formed on the surface of the bilayers. The anchoring strength of this physisorbed layer was found to increase significantly with the size of the hydrophobic block of the polymer, and was strongest for the more hydrophobic polymer, F127. Above the CAC, a dense polymer layer, exhibiting gel-like properties, was found to rapidly grow on the bilayers even after mechanical disruption. The cohesive interaction maintaining the gel layer structure was found to be stronger for F127, and was also found to promote the formation of highly structured aggregates on the bilayers.

  6. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes

    SciTech Connect

    Camley, Brian A.; Lerner, Michael G.; Pastor, Richard W.; Brown, Frank L. H.

    2015-12-28

    The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.

  7. Fabrication and photoelectric properties of self-assembled bilayer lipid membranes on conducting glass.

    PubMed

    Gao, H; Luo, G A; Feng, J; Ottova, A L; Tien, H T

    2000-12-01

    Supported bilayer lipid membranes (s-BLMs with and without the doping of fullerene C60) self-assembled on indium-tin oxide (ITO) glass were fabricated and characterized by cyclic voltammetry and electrochemical impedance spectroscopy using a three-electrode system. The photoelectric properties of the ITO supported planar lipid bilayers were studied. Light intensity of irradiation, bias voltage, and concentration of donors have been found to be limiting factors of the transmembrane photocurrent. The facilitation effect of C60 doping in s-BLMs on the photoinduced electron transfer across s-BLM is discussed. This novel self-assembled ITO/s-BLM system may provide a simple and mechanically stable model for the study of the photoelectric and photodynamic properties of biomembranes.

  8. Polarization-dependent fluorescence of proteins bound to nanopore-confined lipid bilayers

    NASA Astrophysics Data System (ADS)

    Li, R.-Q.; Marek, A.; Smirnov, Alex I.; Grebel, H.

    2008-09-01

    Lipid bilayers are essential structural component of biological membranes of all the living species: from viruses and bacteria to plants and humans. Biophysical and biochemical properties of such membranes are important for understanding physical mechanisms responsible for drug targeting. Binding events between proteins and the membrane may be ascertained by introducing fluorescence markers (chromophores) to the proteins. Here we describe a novel biosensing platform designed to enhance signals of these fluorescence markers. Nanoporous aluminum oxide membranes with and without gold (Au) surface coating have been employed for optical detection of bound conjugated streptavidin to biotinylated lipid bilayers-a model system that mimics protein docking to the membrane surface. Unexpectedly, it was found that fluorescence signals from such structures vary when pumped with E-polarized and H-polarized incident optical beams. The origin of the observed polarization-dependent effects and the implications for enhanced fluorescence detection in a biochip format are being discussed.

  9. Measuring Transmembrane Helix Interaction Strengths in Lipid Bilayers Using Steric Trapping

    PubMed Central

    Hong, Heedeok; Chang, Yu-Chu; Bowie, James U.

    2016-01-01

    We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (Kd,dimer) over a range of six orders of magnitudes. The Kd,dimer limit of measurable target interaction is extended 3–4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer. PMID:23975771

  10. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes.

    PubMed

    Camley, Brian A; Lerner, Michael G; Pastor, Richard W; Brown, Frank L H

    2015-12-28

    The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.

  11. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Camley, Brian A.; Lerner, Michael G.; Pastor, Richard W.; Brown, Frank L. H.

    2015-12-01

    The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.

  12. Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis.

    PubMed

    Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu; Sugiura, Ryuta; Sasaki, Nobuaki; Misawa, Nobuo; Tero, Ryugo; Urisu, Tsuneo; Gardiner, Alastair T; Cogdell, Richard J; Hashimoto, Hideki; Nango, Mamoru

    2011-07-11

    The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

  13. Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro.

    PubMed

    Boggs, Joan M; Rangaraj, Godha; Dicko, Awa

    2012-09-01

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.

  14. A scanning calorimetric study of small molecule-lipid bilayer mixtures.

    PubMed Central

    Sturtevant, J M

    1982-01-01

    The influence of small molecules on the main phase transition of dimyristoyl phosphatidylcholine was observed by high-sensitivity differential scanning calorimetry. The variation with temperature of the excess heat capacity is markedly affected by solid solution formation, which appears to be of frequent occurrence in solutions in lipid bilayers. The experimental results are in some cases in reasonable agreement with ideal solution theory. PMID:6955783

  15. Spherical nanoparticle supported lipid bilayers for the structural study of membrane geometry-sensitive molecules.

    PubMed

    Fu, Riqiang; Gill, Richard L; Kim, Edward Y; Briley, Nicole E; Tyndall, Erin R; Xu, Jie; Li, Conggang; Ramamurthi, Kumaran S; Flanagan, John M; Tian, Fang

    2015-11-11

    Many essential cellular processes including endocytosis and vesicle trafficking require alteration of membrane geometry. These changes are usually mediated by proteins that can sense and/or induce membrane curvature. Using spherical nanoparticle supported lipid bilayers (SSLBs), we characterize how SpoVM, a bacterial development factor, interacts with differently curved membranes by magic angle spinning solid-state NMR. Our results demonstrate that SSLBs are an effective system for structural and topological studies of membrane geometry-sensitive molecules.

  16. Lipid-Protein Correlations in Nanoscale Phospholipid Bilayers by Solid-State NMR

    PubMed Central

    Kijac, Aleksandra; Shih, Amy Y.; Nieuwkoop, Andrew J.; Schulten, Klaus; Sligar, Stephen G.; Rienstra, Chad M.

    2011-01-01

    Nanodiscs are an example of discoidal nanoscale lipid/protein particles that have been extremely useful for the biochemical and biophysical characterization of membrane proteins. They are discoidal lipid bilayer fragments encircled and stabilized by two amphipathic helical proteins named membrane scaffolding protein (MSP), ~10 nm in size. Nanodiscs are homogeneous, easily prepared with reproducible success, amenable to preparations with a variety of lipids, and stable under a range of temperatures. Here we present solid-state NMR (SSNMR) studies on lyophilized, rehydrated POPC Nanodiscs prepared with uniformly 13C, 15N-labeled MSP1D1 (Δ1-11 truncated MSP). Under these conditions, by SSNMR we directly determine the gel-to-liquid crystal lipid phase transition to be at 3 ± 2 °C. Above this phase transition, the lipid 1H signals have slow transverse relaxation, enabling filtering experiments as previously demonstrated for lipid vesicles. We incorporate this approach into two- and three-dimensional heteronuclear SSNMR experiments to examine the MSP1D1 residues interfacing with the lipid bilayer. These 1H-13C and 1H-13C-13C correlation spectra are used to identify and quantify the number of lipid-correlated and solvent-exposed residues by amino acid type, which furthermore is compared with molecular dynamics studies of MSP1D1 in Nanodiscs. This study demonstrates the utility of SSNMR experiments with Nanodiscs for examining lipid-protein interfaces and has important applications for future structural studies of membrane proteins in physiologically relevant formulations. PMID:20804175

  17. Viscosity heterogeneity inside lipid bilayers of single-component phosphatidylcholine liposomes observed with picosecond time-resolved fluorescence spectroscopy.

    PubMed

    Nojima, Yuki; Iwata, Koichi

    2014-07-24

    A number of biochemical reactions proceed inside biomembranes. Because the rate of a chemical reaction is influenced by chemical properties of the reaction field, it is important to examine the chemical properties inside the biomembranes, or lipid bilayer membranes, for understanding biochemical reactions. In this study, we estimate viscosity inside the lipid bilayers of liposomes with picosecond time-resolved fluorescence spectroscopy. trans-Stilbene is solubilized in the lipid bilayers formed by phosphatidylcholines, DSPC, DOPC, DPPC, DMPC, and DLPC, with 18, 18, 16, 14, and 12 carbon atoms in their alkyl chains, respectively, and egg-PC. Viscosity inside the lipid bilayer is estimated from the photoisomerization rate constant and from the rotational relaxation time of the first excited singlet state of trans-stilbene. The effect of the hydrocarbon chain length and temperature on viscosity is examined. The presence of two solvation environments within the lipid bilayer is indicated from the two independent estimations. One environment is 30 to 290 times more viscous than the other. Even single-component lipid bilayers are likely to have heterogeneous structures.

  18. Stable, ligand-doped, poly(bis-SorbPC) lipid bilayer arrays for protein binding and detection.

    PubMed

    Joubert, James R; Smith, Kathryn A; Johnson, Erin; Keogh, John P; Wysocki, Vicki H; Gale, Bruce K; Conboy, John C; Saavedra, S Scott

    2009-06-01

    A continuous-flow microspotter was used to generate planar arrays of stabilized bilayers composed of the polymerizable lipid bis-SorbPC and dopant lipids bearing ligands for proteins. Fluorescence microscopy was used to determine the uniformity of the bilayers and to detect protein binding. After UV-initiated polymerization, poly(lipid) bilayer microarrays were air-stable. Cholera toxin subunit b (CTb) bound to an array of poly(lipid) bilayers doped with GM(1), and the extent of binding was correlated to the mole percentage of GM(1) in each spot. A poly(lipid) bilayer array composed of spots doped with GM(1) and spots doped with biotin-DOPE specifically bound CTb and streptavidin to the respective spots from a dissolved mixture of the two proteins. Poly(bis-SorbPC)/GM(1) arrays retained specific CTb binding capacity after multiple regenerations with a protein denaturing solution and also after exposure to air. In addition, these arrays are stable in vacuum, which allows the use of MALDI-TOF mass spectrometry to detect specifically bound CTb. This work demonstrates the considerable potential of poly(lipid) bilayer arrays for high-throughput binding assays and lipidomics studies. PMID:20355927

  19. Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

    PubMed Central

    Hanson, Sonya M.; Newstead, Simon; Swartz, Kenton J.; Sansom, Mark S.P.

    2015-01-01

    Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1–S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1–S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1–S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular

  20. Capsaicin interaction with TRPV1 channels in a lipid bilayer: molecular dynamics simulation.

    PubMed

    Hanson, Sonya M; Newstead, Simon; Swartz, Kenton J; Sansom, Mark S P

    2015-03-24

    Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1-S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1-S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1-S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular

  1. Super resolution microscopy of lipid bilayer phases and single molecule kinetic studies on merocyanine 540 bound lipid vesicles

    NASA Astrophysics Data System (ADS)

    Kuo, Chin-Kuei

    Recently, observing biological process and structural details in live cell became feasible after the introduction of super-resolution microscopy. Super-resolution microscopy by single molecule localization is the method that has commonly been used for such purpose. There are mainly three approaches to it: stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and point accumulation in nanoscale topology (PAINT). STORM and PALM rely on external laser control and use of photoactivable fluorescent protein or photoswitchable dyes and are technically challenging. The PAINT method relies on the control of thermal reaction rates to enable the switching between bright and dark states. Therefore, many conventional fluorescent probes can be applied in PAINT method and the images denote different information composed of interactions between the probe and its immediate environment by variations of probe parameters. The existence of lipid rafts has been under debates for decades due to the lack of a tool to directly visualize them in live cells. In the thesis, we combine PAINT with a phase sensitive dye, Merocyanine 540, to enable nanoscale observation of phase separation on supported lipid bilayers of mixed liquid/gel phases. The imaging results are presented in the chapter 3. Given that this is the first example of visualization of nanoscale phase separation of lipid bilayers using an optical microscope, we further looked into the kinetics of MC540 monomer dimer equilibrium in lipid bilayers using single molecule intensity time trajectory analysis and polarization dependent imaging. Our finding confirms that perpendicular monomeric MC540 (to the membrance surface) is the emitting speices in our system and it stays fluorescent for roughly 3 ms before it switches off to dark states. This part of analysis is presented in the chapter 4. All the materials, procedures to carry out experiments and data analysis, methods involved in our

  2. Ninety-six-well planar lipid bilayer chip for ion channel recording fabricated by hybrid stereolithography.

    PubMed

    Suzuki, Hiroaki; Le Pioufle, Bruno; Takeuchi, Shoji

    2009-02-01

    We present a micro fluidic chip for parallel ion channel recording in a large array of artificial planar lipid bilayer membranes. To realize a composite structure that features an array of recording wells with free-standing microapertures for lipid bilayer reconstitution, the device was fabricated by the hybrid stereolithography technology, in which a Parylene film with pre-formed microapertures was inserted during the rapid stereolithography process. We designed and tested a hybrid chip that has 96 (12x8) addressable recording wells to demonstrate recording of ion channel current in high-throughput manner. Measurement was done by sequentially moving the recording electrode, and, as a result, the channel current of model membrane protein was detected in 44 wells out of 96. We also showed that this hybrid fabrication process was capable of integrating micropatterned electrodes suitable for automated recording. These results support the efficiency of our present architecture of the parallel ion channel recording chip toward realization of the high-throughput screening of ion channel proteins in the artificial lipid bilayer system.

  3. Bilayer registry in a multicomponent asymmetric membrane: Dependence on lipid composition and chain length

    SciTech Connect

    Polley, Anirban; Mayor, Satyajit; Rao, Madan E-mail: madan@ncbs.res.in

    2014-08-14

    A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component asymmetric lipid bilayer comprising an unsaturated palmitoyl-oleoyl-phosphatidyl-choline, a saturated sphingomyelin, and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered (l{sub o})-liquid disordered (l{sub d}) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed l{sub d} phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the l{sub o} domains across the two leaflets, resulting in bilayer registry. This transbilayer correlation depends sensitively upon the chain length of the participating lipids and possibly other features of lipid chemistry, such as degree of saturation. We find that the l{sub o} domains in the upper leaflet can induce phase segregation in the lower leaflet, when the latter is nominally in the mixed (l{sub d}) phase.

  4. pH regulation of amphotericin B channels activity in the bilayer lipid membrane

    PubMed Central

    Shahmoradi, Tahereh; Sepehry, Hamid; Ashrafpour, Manuchehr

    2016-01-01

    Background: Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of systemic fungal infections in spite of its secondary effects. The pH plays a crucial role in modulating biophysical features of ion channels in the bilayer lipid membranes. Aim: In this study, the role of pH in the regulation of AmB channel was assessed by single channel recording of ion channel incorporated in the artificial membrane. Materials and Methods: Bilayer lipid membrane was formed by phosphatidylcholine in a 350 μm diameter aperture between two chambers, cis and trans contained 200/50 mMKCl solutions, respectively; then AmB was incorporated into the bilayer lipid membrane. Single channel recordings were used to indicate the effects of pH changes on AmB channels activity. The records were analyzed by Clamp fit 10 software. Results: A kinetic analysis of single channel currents indicated a cation ion channel with 500 pS conductance and voltage-dependence of the open probability of the AmB channel (Po). A reduction of cis pH to 6 decreased Po and conductance. This effect was also voltage-dependent, being greater at a more positive above −40. The pH changes in the range of 6-8 had no effect on the reversal potential and ion selectivity. Conclusion: Our data indicated that extracellular acidity can reduce AmB activity. PMID:27003977

  5. Protocells: Modular Mesoporous Silica Nanoparticle-Supported Lipid Bilayers for Drug Delivery.

    PubMed

    Butler, Kimberly S; Durfee, Paul N; Theron, Christophe; Ashley, Carlee E; Carnes, Eric C; Brinker, C Jeffrey

    2016-04-27

    Mesoporous silica nanoparticle-supported lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicle. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core has variable size and shape to direct biodistribution and a controlled pore size and surface chemistry to accommodate diverse cargo. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. This review describes the individual components of the platform, including the mesoporous silica nanoparticle core and supported lipid bilayer, their assembly (by multiple techniques) into a protocell, and the combined, often synergistic, performance of the protocell based on in vitro and in vivo studies, including the assessment of biocompatibility and toxicity. In closing, the many emerging variations of the protocell theme and the future directions for protocell research are commented on. PMID:26780591

  6. Protocells: Modular Mesoporous Silica Nanoparticle-Supported Lipid Bilayers for Drug Delivery.

    PubMed

    Butler, Kimberly S; Durfee, Paul N; Theron, Christophe; Ashley, Carlee E; Carnes, Eric C; Brinker, C Jeffrey

    2016-04-27

    Mesoporous silica nanoparticle-supported lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicle. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core has variable size and shape to direct biodistribution and a controlled pore size and surface chemistry to accommodate diverse cargo. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. This review describes the individual components of the platform, including the mesoporous silica nanoparticle core and supported lipid bilayer, their assembly (by multiple techniques) into a protocell, and the combined, often synergistic, performance of the protocell based on in vitro and in vivo studies, including the assessment of biocompatibility and toxicity. In closing, the many emerging variations of the protocell theme and the future directions for protocell research are commented on.

  7. Biopolymer-Lipid Bilayer Interaction Modulates the Physical Properties of Liposomes: Mechanism and Structure.

    PubMed

    Tan, Chen; Zhang, Yating; Abbas, Shabbar; Feng, Biao; Zhang, Xiaoming; Xia, Wenshui; Xia, Shuqin

    2015-08-19

    This study was conducted to elucidate the conformational dependence of the modulating ability of chitosan, a positively charged biopolymer, on a new type of liposome composed of mixed lipids including egg yolk phosphatidylcholine (EYPC) and nonionic surfactant (Tween 80). Analysis of the dynamic and structure of bilayer membrane upon interaction with chitosan by fluorescence and electron paramagnetic resonance techniques demonstrated that, in addition to providing a physical barrier for the membrane surface, the adsorption of chitosan extended and crimped chains rigidified the lipid membrane. However, the decrease in relative microviscosity and order parameter suggested that the presence of chitosan coils disturbed the membrane organization. It was also noted that the increase of fluidity in the lipid bilayer center was not pronounced, indicating the shallow penetration of coils into the hydrophobic interior of bilayer. Microscopic observations revealed that chitosan adsorption not only affected the morphology of liposomes but also modulated the particle aggregation and fusion. Especially, a number of very heterogeneous particles were visualized, which tended to confirm the role of chitosan coils as a "polymeric surfactant". In addition to particle deformation, the membrane permeability was also tuned. These findings may provide a new perspective to understand the physiological functionality of biopolymer and design biopolymer-liposome composite structures as delivery systems for bioactive components. PMID:26173584

  8. Direct Measurement of the Mechanical Properties of Lipid Phases in Supported Bilayers

    PubMed Central

    Picas, Laura; Rico, Felix; Scheuring, Simon

    2012-01-01

    Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ∼200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (kA) as 106 pN/nm and 199 pN/nm and the bending stiffness (kc) as 18 kBT and 57 kBT for the liquid and gel phases, respectively. PMID:22225813

  9. Experimental Determination of High-Order Bending Elastic Constants of Lipid Bilayers.

    PubMed

    Toscano-Flores, Liliana G; Jacinto-Méndez, Damián; Carbajal-Tinoco, Mauricio D

    2016-06-30

    We present a method to describe the formation of small lipid vesicles in terms of three bending elastic constants that can be experimentally measured. Our method combines a general expression of the elastic free energy of the bilayer and the thermodynamic description of molecular aggregation. The resulting model requires the size distribution of liposomes, which is determined from the X-ray scattered intensity spectra of vesicular dispersions. By using two different preparation methods, we studied a series of vesicular solutions made of distinct lipids and we obtained their corresponding bending elastic constants that are consistent with known bending rigidities. PMID:27267752

  10. Incorporation of a calixarene-based glucose functionalised bolaamphiphile into lipid bilayers for multivalent lectin recognition.

    PubMed

    Aleandri, S; Casnati, A; Fantuzzi, L; Mancini, G; Rispoli, G; Sansone, F

    2013-08-01

    The synthesis, characterization and inclusion in liposomes of a glucosylated bolaamphiphile built on a calix[4]arene scaffold are described. The new glucocalixarene bolaamphiphile destabilizes bilayers of saturated lipids whereas it rigidifies those of unsaturated lipids, thus reducing leakage of calcein from the liposome internal aqueous compartment. Moreover, from fluorescence and turbidimetry experiments it was found that the glucose units of bolaamphiphile 1 functionalised liposomes allow a specific multivalent interaction with the tetrameric glucose binding protein Concanavalin A. These results therefore represent a novel strategy to functionalise liposomes with saccharides, exploiting multivalent glycosylated ligands to be used in the preparation of drug delivery systems potentially able to target specific lectins. PMID:23771589

  11. Molecular Dynamics Study of Gramicidin A in Lipid Bilayer: Electrostatic Map and Ion Conduction

    NASA Astrophysics Data System (ADS)

    Saito, Hiroaki; Iwayama, Masashi; Kawaguchi, Kazutomo; Mizukami, Taku; Miyakawa, Takeshi; Takasu, Masako; Nagao, Hidemi

    The electrostatic potential (ESP) of gramicidin A (GA) in the DMPC lipid bilayers with/without an external uniform electrostatic field was investigated by molecular dynamics (MD) simulation. We found that the ESP profile with an external electrostatic field became step shape. The water and polar groups of the lipid and GA are rearranged in order to restore a flat ESP in the water bulk and GA channel interior. The reorientation of the polar head group enhances the ESP difference between each hydration regions of the membrane, and this should yield an increase of ion conductance through the GA channel.

  12. Neutron reflectivity studies of single lipid bilayers supported on planar substrates

    SciTech Connect

    Krueger, S.; Orts, W.J.; Berk, N.F.; Majkrzak, C.F.; Koenig, B.W.

    1994-12-31

    Neutron reflectivity was used to probe the structure of single phosphatidylcholine (PC) lipid bilayers adsorbed onto a planar silicon surface in an aqueous environment. Fluctuations in the neutron scattering length density profiles perpendicular to the silicon/water interface were determined for different lipids as a function of the hydrocarbon chain length. The lipids were studied in both the gel and liquid crystalline phases by monitoring changes in the specularly-reflected neutron intensity as a function of temperature. Contrast variation of the neutron scattering length density was applied to both the lipid and the solvent. Scattering length density profiles were determined using both model-independent and model-dependent fitting methods. During the reflectivity measurements, a novel experimental set-up was implemented to decrease the incoherent background scattering due to the solvent. Thus, the reflectivity was measured to Q {approx} 0.3{Angstrom}{sup -1}, covering up to seven orders of magnitude in reflected intensity, for PC bilayers in D{sub 2}O and silicon-matched (38% D{sub 2}O/62% H{sub 2}O) water. The kinetics of lipid adsorption at the silicon/water interface were also explored by observing changes in the reflectivity at low Q values under silicon-matched water conditions.

  13. Flexible-to-semiflexible chain crossover on the pressure-area isotherm of a lipid bilayer

    NASA Astrophysics Data System (ADS)

    Krivonos, I. N.; Mukhin, S. I.

    2008-01-01

    We find theoretically that competition between ˜ K f q 4 and ˜ Qq 2 terms in the Fourier-transformed conformational energy of a single-lipid chain, in combination with interchain entropic repulsion in the hydrophobic part of the lipid (bi)layer, may cause a crossover on the bilayer pressure-area isotherm P( A)˜( A- A 0)-α. The crossover manifests itself in the transition from α = 5/3 to α = 3. Our microscopic model represents a single-lipid molecule as a worm-like chain with a finite irreducible cross-section area A 0, a flexural rigidity K f , and a stretching modulus Q in a parabolic potential with the self-consistent curvature B( A) formed by entropic interactions between hydrocarbon chains in the lipid layer. The crossover area A* obeys the relation Q/√ K f B( A*) ≈ 2. We predict a peculiar possibility of deducing the effective elastic moduli K f and Q of an individual hydrocarbon chain from the analysis of the isotherm with such a crossover. Also calculated is the crossover-related behavior of the area compressibility modulus K A , the equilibrium area per lipid A t , and the chain order parameter S(θ).

  14. Molecular dynamics study of electrostatic potential along lipid bilayer with gramicidin A

    NASA Astrophysics Data System (ADS)

    Saito, Hiroaki; Nishimura, Megumi; Takagi, Hiroyuki; Miyakawa, Takeshi; Kawaguchi, Kazutomo; Nagao, Hidemi

    2013-02-01

    The structure and electrostatic potential profile of the DMPC lipid bilayers with a gramicidin A (GA) were studied by molecular dynamics (MD) simulation. The MD simulation reproduced the effect of GA on the membrane structure; the area per lipid decreases and membrane thickness increases, and the observed membrane structures correspond to the experimental data. The polar headgroup of lipid was found to orient toward the membrane normal as the lipid approaches the GA. The observed electrostatic potential map showed that the electrostatic potential around the region of GA gate was lower than the others at the same level of the membrane normal and the values of electrostatic potential in the pore region of GA were negative. These results indicate that a cation in the aqueous region of membrane can be electrostatically led to the GA entrance and penetrate the GA channel following the gradient of ion concentration.

  15. Spontaneous curvature of bilayer membranes from molecular simulations: Asymmetric lipid densities and asymmetric adsorption

    NASA Astrophysics Data System (ADS)

    RóŻycki, Bartosz; Lipowsky, Reinhard

    2015-02-01

    Biomimetic and biological membranes consist of molecular bilayers with two leaflets which are typically exposed to different aqueous environments and may differ in their molecular density or composition. Because of these asymmetries, the membranes prefer to curve in a certain manner as quantitatively described by their spontaneous curvature. Here, we study such asymmetric membranes via coarse-grained molecular dynamics simulations. We consider two mechanisms for the generation of spontaneous curvature: (i) different lipid densities within the two leaflets and (ii) leaflets exposed to different concentrations of adsorbing particles. We focus on membranes that experience no mechanical tension and describe two methods to compute the spontaneous curvature. The first method is based on the detailed structure of the bilayer's stress profile which can hardly be measured experimentally. The other method starts from the intuitive view that the bilayer represents a thin fluid film bounded by two interfaces and reduces the complexity of the stress profile to a few membrane parameters that can be measured experimentally. For the case of asymmetric adsorption, we introduce a simulation protocol based on two bilayers separated by two aqueous compartments with different adsorbate concentrations. The adsorption of small particles with a size below 1 nm is shown to generate large spontaneous curvatures up to about 1/(24 nm). Our computational approach is quite general: it can be applied to any molecular model of bilayer membranes and can be extended to other mechanisms for the generation of spontaneous curvatures as provided, e.g., by asymmetric lipid composition or depletion layers of solute molecules.

  16. The dependence of the lipid bilayer membrane: buffer partition coefficient of pentobarbitone on pH and lipid composition.

    PubMed Central

    Miller, K W; Yu, S C

    1977-01-01

    1 The membrane/buffer partition coefficient of [14C]-pentobarbitone has been determined as a function of the lipid composition of bilayer membranes. 2 A new technique based on ultrafiltration gave comparable results to conventional techniques but required less time for equilbration. 3 The membrane/buffer coefficient was independent of pentobarbitone concentration in the range studies. 4 The apparent partition coefficient varied with pH and was a linear function of the degree of dissociation of pentobarbition. 5 Both the charged and uncharged forms of pentobarbitone partitioned into the membrane, the latter to a much greater extent than the former. 6 At low pH the highest partition coefficient observed was in egg phosphatidylcholine bilayer membranes. 7 Incorporation of cholesterol or phosphatidic acid into phosphatidylcholine membranes greatly reduced the partition coefficient. 8 High pressures do not greatly change these partition coefficients. PMID:21013

  17. Simulated Permeation and Characterization of PEGylated Gold Nanoparticles in a Lipid Bilayer System.

    PubMed

    Oroskar, Priyanka A; Jameson, Cynthia J; Murad, Sohail

    2016-08-01

    PEGylated gold nanoparticles are considered suitable nanocarriers for use in biomedical applications and targeted drug delivery systems. In our previous investigation with the alkanethiol-functionalized gold nanoparticle, we found that permeation across a protein-free phospholipid membrane resulted in damaging effects of lipid displacement and water and ion leakage. In the present study, we carry out a series of coarse-grained molecular simulations to explore permeation of lipid bilayer systems by a PEGylated gold nanoparticle, especially at the bulk-liquid-lipid interface as well as the interface between the two lipid leaflets. Initially, we examine molecular-level details of a PEGylated gold nanoparticle (constructed from cycled annealing) in water and find a distribution of ligand configurations (from mushroom to brush states) present in nanoparticles with medium to high surface coverage. We also find that the characteristic properties of the PEGylated gold nanoparticle do not change when it is placed in a salt solution. In our permeation studies, we investigate events of water and ion penetration as well as lipid translocation while varying the ligand length, nanoparticle surface coverage, and ion concentration gradient of our system. Results from our studies show the following: (1) The number of water molecules in the interior of the membrane during ligand-coated nanoparticle permeation increases with PEGn-SH surface coverage, ligand length, and permeation velocity but is not sensitive to the ion concentration gradient. (2) Lipid molecules do not leave the membrane; instead they complete trans-bilayer lipid flip-flop with longer ligands and higher surface coverages. (3) The lack of formation of stable water pores prevents ion translocation. (4) The PEGylated nanoparticle causes less damage to the membrane overall due to favorable interactions with the lipid headgroups which may explain why experimentalists observe endocytosis of PEGylated nanocarriers in vivo.

  18. Simulated Permeation and Characterization of PEGylated Gold Nanoparticles in a Lipid Bilayer System.

    PubMed

    Oroskar, Priyanka A; Jameson, Cynthia J; Murad, Sohail

    2016-08-01

    PEGylated gold nanoparticles are considered suitable nanocarriers for use in biomedical applications and targeted drug delivery systems. In our previous investigation with the alkanethiol-functionalized gold nanoparticle, we found that permeation across a protein-free phospholipid membrane resulted in damaging effects of lipid displacement and water and ion leakage. In the present study, we carry out a series of coarse-grained molecular simulations to explore permeation of lipid bilayer systems by a PEGylated gold nanoparticle, especially at the bulk-liquid-lipid interface as well as the interface between the two lipid leaflets. Initially, we examine molecular-level details of a PEGylated gold nanoparticle (constructed from cycled annealing) in water and find a distribution of ligand configurations (from mushroom to brush states) present in nanoparticles with medium to high surface coverage. We also find that the characteristic properties of the PEGylated gold nanoparticle do not change when it is placed in a salt solution. In our permeation studies, we investigate events of water and ion penetration as well as lipid translocation while varying the ligand length, nanoparticle surface coverage, and ion concentration gradient of our system. Results from our studies show the following: (1) The number of water molecules in the interior of the membrane during ligand-coated nanoparticle permeation increases with PEGn-SH surface coverage, ligand length, and permeation velocity but is not sensitive to the ion concentration gradient. (2) Lipid molecules do not leave the membrane; instead they complete trans-bilayer lipid flip-flop with longer ligands and higher surface coverages. (3) The lack of formation of stable water pores prevents ion translocation. (4) The PEGylated nanoparticle causes less damage to the membrane overall due to favorable interactions with the lipid headgroups which may explain why experimentalists observe endocytosis of PEGylated nanocarriers in vivo

  19. Influence of lipid bilayer properties on nanodisc formation mediated by styrene/maleic acid copolymers.

    PubMed

    Cuevas Arenas, Rodrigo; Klingler, Johannes; Vargas, Carolyn; Keller, Sandro

    2016-08-11

    Copolymers of styrene and maleic acid (SMA) have gained great attention as alternatives to conventional detergents, as they offer decisive advantages for studying membrane proteins and lipids in vitro. These polymers self-insert into artificial and biological membranes and, at sufficiently high concentrations, solubilise them into disc-shaped nanostructures containing a lipid bilayer core surrounded by a polymer belt. We have used (31)P nuclear magnetic resonance spectroscopy and dynamic light scattering to systematically study the solubilisation of vesicles composed of saturated or unsaturated phospholipids by an SMA copolymer with a 3 : 1 styrene/maleic acid molar ratio at different temperatures. Solubilisation was thermodynamically rationalised in terms of a three-stage model that treats various lipid/polymer aggregates as pseudophases. The solubilising capacity of SMA(3 : 1) towards a saturated lipid is higher in the gel than in the liquid-crystalline state of the membrane even though solubilisation is slower. Although the solubilisation of mixed fluid membranes is non-selective, the presence of a non-bilayer phospholipid lowers the threshold at which the membrane becomes saturated with SMA(3 : 1) but raises the polymer concentration required for complete solubilisation. Both of these trends can be explained by considering the vesicle-to-nanodisc transfer free energies of the lipid and the polymer. On the basis of the phase diagrams thus obtained, re-association of polymer-solubilised lipids with vesicles is possible under mild conditions, which has implications for the reconstitution of proteins and lipids from nanodiscs into vesicular membranes. Finally, the phase diagrams provide evidence for the absence of free SMA(3 : 1) in vesicular lipid suspensions. PMID:27471007

  20. Ionizable Nitroxides for Studying Local Electrostatic Properties of Lipid Bilayers and Protein Systems by EPR

    PubMed Central

    Voinov, Maxim A.; Smirnov, Alex I.

    2016-01-01

    Electrostatic interactions are known to play one of the major roles in the myriad of biochemical and biophysical processes. In this Chapter we describe biophysical methods to probe local electrostatic potentials of proteins and lipid bilayer systems that is based on an observation of reversible protonation of nitroxides by EPR. Two types of the electrostatic probes are discussed. The first one includes methanethiosulfonate derivatives of protonatable nitroxides that could be used for highly specific covalent modification of the cysteine’s sulfhydryl groups. Such spin labels are very similar in magnetic parameters and chemical properties to conventional MTSL making them suitable for studying local electrostatic properties of protein-lipid interfaces. The second type of EPR probes is designed as spin-labeled phospholipids having a protonatable nitroxide tethered to the polar head group. The probes of both types report on their ionization state through changes in magnetic parameters and a degree of rotational averaging, thus, allowing one to determine the electrostatic contribution to the interfacial pKa of the nitroxide, and, therefore, determining the local electrostatic potential. Due to their small molecular volume these probes cause a minimal perturbation to the protein or lipid system while covalent attachment secure the position of the reporter nitroxides. Experimental procedures to characterize and calibrate these probes by EPR and also the methods to analyze the EPR spectra by least-squares simulations are also outlined. The ionizable nitroxide labels and the nitroxide-labeled phospholipids described so far cover an exceptionally wide pH range from ca. 2.5 to 7.0 pH units making them suitable to study a broad range of biophysical phenomena especially at the negatively charged lipid bilayer surfaces. The rationale for selecting proper electrostatically neutral interface for calibrating such probes and example of studying surface potential of lipid bilayer is

  1. Feeling the hidden mechanical forces in lipid bilayer is an original sense

    PubMed Central

    Anishkin, Andriy; Loukin, Stephen H.; Teng, Jinfeng; Kung, Ching

    2014-01-01

    Life’s origin entails enclosing a compartment to hoard material, energy, and information. The envelope necessarily comprises amphipaths, such as prebiotic fatty acids, to partition the two aqueous domains. The self-assembled lipid bilayer comes with a set of properties including its strong anisotropic internal forces that are chemically or physically malleable. Added bilayer stretch can alter force vectors on embedded proteins to effect conformational change. The force-from-lipid principle was demonstrated 25 y ago when stretches opened purified Escherichia coli MscL channels reconstituted into artificial bilayers. This reductionistic exercise has rigorously been recapitulated recently with two vertebrate mechanosensitive K+ channels (TREK1 and TRAAK). Membrane stretches have also been known to activate various voltage-, ligand-, or Ca2+-gated channels. Careful analyses showed that Kv, the canonical voltage-gated channel, is in fact exquisitely sensitive even to very small tension. In an unexpected context, the canonical transient-receptor-potential channels in the Drosophila eye, long presumed to open by ligand binding, is apparently opened by membrane force due to PIP2 hydrolysis-induced changes in bilayer strain. Being the intimate medium, lipids govern membrane proteins by physics as well as chemistry. This principle should not be a surprise because it parallels water’s paramount role in the structure and function of soluble proteins. Today, overt or covert mechanical forces govern cell biological processes and produce sensations. At the genesis, a bilayer’s response to osmotic force is likely among the first senses to deal with the capricious primordial sea. PMID:24850861

  2. Lipid Bilayers: The Effect of Force Field on Ordering and Dynamics.

    PubMed

    Poger, David; Mark, Alan E

    2012-11-13

    The sensitivity of the structure and dynamics of a fully hydrated pure bilayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in molecular dynamics simulations to changes in force-field and simulation parameters has been assessed. Three related force fields (the Gromos 54A7 force field, a Gromos 53A6-derived parameter set and a variant of the Berger parameters) in combination with either particle-mesh Ewald (PME) or a reaction field (RF) were compared. Structural properties such as the area per lipid, carbon-deuterium order parameters, electron density profile and bilayer thicknesses, are reproduced by all the parameter sets within the uncertainty of the available experimental data. However, there are clear differences in the ordering of the glycerol backbone and choline headgroup, and the orientation of the headgroup dipole. In some cases, the degree of ordering was reminiscent of a liquid-ordered phase. It is also shown that, although the lateral diffusion of the lipids in the plane of the bilayer is often used to validate lipid force fields, because of the uncertainty in the experimental measurements and the fact that the lateral diffusion is dependent on the choice of the simulation conditions, it should not be employed as a measure of quality. Finally, the simulations show that the effect of small changes in force-field parameters on the structure and dynamics of a bilayer is more significant than the treatment of the long-range electrostatic interactions using RF or PME. Overall, the Gromos 54A7 best reproduced the range of experimental data examined. PMID:26605633

  3. Effects of truncating van der Waals interactions in lipid bilayer simulations

    NASA Astrophysics Data System (ADS)

    Huang, Kun; García, Angel E.

    2014-09-01

    In membrane simulations, it is known that truncating electrostatic interactions results in artificial ordering of lipids at the truncation distance. However, less attention has been paid to the effect of truncating van der Waals (VDW) interactions. Since the VDW potential decays as r-6, it is frequently neglected beyond a cutoff of around 1 nm. In some cases, analytical dispersion corrections appropriate for isotropic systems are applied to the pressure and the potential energy. In this work, we systematically study the effect of truncating VDW interactions at different cutoffs in 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine bilayers with the Berger force field. We show that the area per lipid decreases systematically when the VDW cutoff (rc) increases. This dependence persists even when dispersion corrections are applied. Since the analytical form of the dispersion correction is only appropriate for isotropic systems, we suggest that a long VDW cutoff should be used in preference over a short VDW cutoff. To determine the appropriate cutoff, we simulate liquid pentadecane with the Berger parameters and find that rc ≥ 1.4 nm is sufficient to reproduce the density and the heat of vaporization of pentadecane. Bilayers simulated with rc ≥ 1.4 nm show an improved agreement with experiments in both the form factors and the deuterium order parameters. Finally, we report that the VDW cutoff has a significant impact on the lipid flip-flop energetics and an inappropriate short VDW cutoff results in a bilayer that is prone to form water defects across the bilayer.

  4. Effects of truncating van der Waals interactions in lipid bilayer simulations

    SciTech Connect

    Huang, Kun; García, Angel E.

    2014-09-14

    In membrane simulations, it is known that truncating electrostatic interactions results in artificial ordering of lipids at the truncation distance. However, less attention has been paid to the effect of truncating van der Waals (VDW) interactions. Since the VDW potential decays as r{sup −6}, it is frequently neglected beyond a cutoff of around 1 nm. In some cases, analytical dispersion corrections appropriate for isotropic systems are applied to the pressure and the potential energy. In this work, we systematically study the effect of truncating VDW interactions at different cutoffs in 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine bilayers with the Berger force field. We show that the area per lipid decreases systematically when the VDW cutoff (r{sub c}) increases. This dependence persists even when dispersion corrections are applied. Since the analytical form of the dispersion correction is only appropriate for isotropic systems, we suggest that a long VDW cutoff should be used in preference over a short VDW cutoff. To determine the appropriate cutoff, we simulate liquid pentadecane with the Berger parameters and find that r{sub c} ≥ 1.4 nm is sufficient to reproduce the density and the heat of vaporization of pentadecane. Bilayers simulated with r{sub c} ≥ 1.4 nm show an improved agreement with experiments in both the form factors and the deuterium order parameters. Finally, we report that the VDW cutoff has a significant impact on the lipid flip-flop energetics and an inappropriate short VDW cutoff results in a bilayer that is prone to form water defects across the bilayer.

  5. Raft Formation in Lipid Bilayers Coupled to Curvature

    PubMed Central

    Sadeghi, Sina; Müller, Marcus; Vink, Richard L.C.

    2014-01-01

    We present computer simulations of a membrane in which the local composition is coupled to the local membrane curvature. At high temperatures (i.e., above the temperature of macroscopic phase separation), finite-sized transient domains are observed, reminiscent of lipid rafts. The domain size is in the range of hundred nanometers, and set by the membrane elastic properties. These findings are in line with the notion of the membrane as a curvature-induced microemulsion. At low temperature, the membrane phase separates. The transition to the phase-separated regime is continuous and belongs to the two-dimensional Ising universality class when the coupling to curvature is weak, but becomes first-order for strong curvature-composition coupling. PMID:25296311

  6. Raft formation in lipid bilayers coupled to curvature.

    PubMed

    Sadeghi, Sina; Müller, Marcus; Vink, Richard L C

    2014-10-01

    We present computer simulations of a membrane in which the local composition is coupled to the local membrane curvature. At high temperatures (i.e., above the temperature of macroscopic phase separation), finite-sized transient domains are observed, reminiscent of lipid rafts. The domain size is in the range of hundred nanometers, and set by the membrane elastic properties. These findings are in line with the notion of the membrane as a curvature-induced microemulsion. At low temperature, the membrane phase separates. The transition to the phase-separated regime is continuous and belongs to the two-dimensional Ising universality class when the coupling to curvature is weak, but becomes first-order for strong curvature-composition coupling.

  7. Small-molecule photostabilizing agents are modifiers of lipid bilayer properties.

    PubMed

    Alejo, Jose L; Blanchard, Scott C; Andersen, Olaf S

    2013-06-01

    Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs--cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)--on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx-the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model

  8. Small-molecule photostabilizing agents are modifiers of lipid bilayer properties.

    PubMed

    Alejo, Jose L; Blanchard, Scott C; Andersen, Olaf S

    2013-06-01

    Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs--cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)--on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx-the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model

  9. Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties

    PubMed Central

    Alejo, Jose L.; Blanchard, Scott C.; Andersen, Olaf S.

    2013-01-01

    Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs—cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)—on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl+). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl+ influx—the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model

  10. Sensitive detection and prevention of protein adsorption on biomaterial (lipid bilayer) surfaces

    NASA Astrophysics Data System (ADS)

    Xu, Zhong

    2000-10-01

    The adsorption of proteins on biomaterial surfaces is recognized as the first and the most important event that determines or directs consequent host responses. The amount, composition, conformation, and binding affinity of the adsorbed proteins are critical in determining events leading to blood coagulation, platelet adhesion and activation, mammalian and bacterial cell adhesion, and complement activation. This thesis is concerned with the non-specific adsorption of plasma proteins on self-assembled phospholipid surfaces. To examine molecular events in detail, adsorption of plasma proteins on lipid bilayers was studied on both supported planar bilayer and liposomes (phospholipid vesicles) systems. Monolayers of mixed distearoylphosphatidlcholine (DSPC) and PEO (MW. 2000)-grafted distearoylphosphatidylethanolanane (PEO2k-DSPE) were deposited on DSPE-coated quartz substrates by Langmuir-Blodgett (LB) deposition technique. A sensitive total internal reflection fluorescence (TIRF) method, capable of detecting the adsorption of <0.3 ng/cm2 of fluorescein-labeled fibrinogen at a temporal resolution of 0.2 sec, was utilized to study adsorption kinetics of plasma proteins on the supported lipid bilayers. All supported lipid bilayers exhibited over a magnitude reduction in adsorbed plasma proteins compared with the quartz substrate. The increase of PEO2k-DSPE density in the mixed bilayers slightly increased the amount of adsorbed proteins on the bilayers. Plasma proteins adsorbed on liposomes (composed of DSPC and cholesterol) were studied using a spin column procedure for liposome isolation, and SDS-polyacrylamide gel electrophoresis and immunoanalysis for protein separation, quantification and identification. Plasma proteins isolated from liposome surfaces showed distinct and complex profiles. Albumin and fibrinogen were identified as two major plasma proteins that undergo non-specific adsorption on liposomes. Incorporating (PEO2k-DSPE) into liposomes reduced the amount

  11. Lipid bilayer elasticity measurements in giant liposomes in contact with a solubilizing surfactant.

    PubMed

    Ménager, Christine; Guemghar, Dihya; Perzynski, Régine; Lesieur, Sylviane; Cabuil, Valérie

    2008-05-01

    A new method to probe the modification of the elasticity of phospholipid bilayers is presented. The purpose here concerns the action of a solubilizing surfactant on a vesicle bilayer. This method is based on the measure of the under-field elongation of giant magnetic-fluid-loaded liposomes. The addition of the nonionic surfactant octyl-beta-d-glucopyranoside (OG) to vesicles at sublytic levels increases the elasticity of the membrane, as shown by the value of the bending modulus K(b), which decreases. K(b) measured around 20 kT for a pure 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer indeed reaches a few kT in the case of the mixed OG-DOPC bilayer. The purpose and interest of this study are to allow the determination of the membrane bending modulus before and after the addition of OG on the same magnetic liposome. Moreover, the experimental conditions used in this work allow the control of lipid and surfactant molar fractions in the mixed aggregates. Then, optical microscopy observation can be performed on samples in well-defined regions of the OG-phospholipid state diagram. PMID:18363418

  12. Evanescent wave cavity ringdown spectroscopy: a platform for the study of supported lipid bilayers.

    PubMed

    Powell, Hayley V; O'Connell, Michael A; Zhang, Meiqin; Mackenzie, Stuart R; Unwin, Patrick R

    2012-03-01

    Evanescent wave cavity ringdown spectroscopy (EW-CRDS) is advocated as an approach for monitoring the formation of supported lipid bilayers (SLBs) on quartz substrates in situ and for the quantitative study of fast molecular adsorption kinetics at the resulting modified biomimetic surface. This approach is illustrated using SLBs of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Complementary atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) measurements confirm the formation of bilayers on quartz. The subsequent interaction of the porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p',p'',p'''-tetrasulfonic acid tetrasodium hydrate (TPPS) with the cationic bilayer-modified silica surface has been studied using EW-CRDS combined with an impinging-jet to deliver analyte to the surface in a well-defined manner. The adsorption of TPPS to the bilayer was kinetically controlled and the adsorption rate constant was found to be 1.7 (±0.6) × 10(-4) cm s(-1) from finite element modeling of the jet hydrodynamics and associated convective-diffusion equation, coupled to a first-order surface process describing adsorption. These proof-of-concept studies provide a platform for the investigation of molecular processes at biomembranes using EW-CRDS for chemical species showing optical absorbance in the visible and ultraviolet range.

  13. Atomic Force Microscopy Studies of Lipophosphoglycan (LPG) Molecules in Lipid Bilayers

    SciTech Connect

    LAST, JULIE A.; HUBER, TINA; SASAKI, DARRYL Y.; SALVATORE, BRIAN; TURCO, SALVATORE J.

    2003-03-01

    Lipophosphoglycan (LPG) is a lypopolysaccharide found on the surface of the parasite Leishmania donovani that is thought to play an essential role in the infection of humans with leishamniasis. LPG acts as an adhesion point for the parasite to the gut of the sand fly, whose bite is responsible for transmitting the disease. In addition, LPG acts to inhibit protein kinase C (PKC) in the human macrophage, possibly by structural changes in the membrane. The Ca{sup 2+} ion is believed to play a role in the infection cycle, acting both as a crosslinker between LPG molecules and by playing a part in modulating PKC activity. To gain insight into the structure of LPG within a supported lipid membrane and into the structural changes that occur due to Ca{sup 2+} ions, we have employed the atomic force microscope (AFM). We have observed that the LPG molecules inhibit bilayer fusion, resulting in bilayer islands on the mica surface. One experiment suggests that the LPG molecules are parallel to the mica surface and that the structure of the LPG changes upon addition of Ca{sup 2+}, with an increase in the height of the LPG molecules from the bilayer surface and an almost complete coverage of LPG on the bilayer island.

  14. Molecular Dynamics Simulation Study of Permeation of Molecules through Skin Lipid Bilayer.

    PubMed

    Gupta, Rakesh; Sridhar, D B; Rai, Beena

    2016-09-01

    Stratum Corneum (SC), the outermost layer of skin, is mainly responsible for skin's barrier function. The complex lipid matrix of SC determines these barrier properties. In this study, the lipid matrix is modeled as an equimolar mixture of ceramide (CER), cholesterol (CHOL), and free fatty acid (FFA). The permeation of water, oxygen, ethanol, acetic acid, urea, butanol, benzene, dimethyl sulfoxide (DMSO), toluene, phenol, styrene, and ethylbenzene across this layer is studied using a constrained MD simulations technique. Several long constrained simulations are performed at a skin temperature of 310 K under NPT conditions. The free energy profiles and diffusion coefficients along the bilayer normal have been calculated for each molecule. Permeability coefficients are also calculated and compared with experimental data. The main resistance for the permeation of hydrophilic and hydrophobic permeants has been found to be in the interior of the lipid bilayer and near the lipid-water interface, respectively. The obtained permeability is found to be a few orders of magnitude higher than experimental values for hydrophilic molecules while for hydrophobic molecules more discrepancy was observed. Overall, the qualitative ranking is consistent with the experiments. PMID:27518707

  15. Oxide and organic nano-porous sol-gel materials synthesized for lipid bilayer formation

    NASA Astrophysics Data System (ADS)

    Nellis, Barbara

    Sol-gel synthesized materials have many applications and in this thesis, different sol-gel films have been investigated as a scaffolding substrate for vesicle-fused lipid bilayers. Oxide, organic, and phosphate-based xerogels have been characterized using methods such as scanning electron microscopy (SEM), x-ray diffraction (XRD), atomic force micrsocopy (AFM) and contact angle goniometry to determine their viability as lipid membrane supports. Membranes containing 1,2-Dioleoyl-Glycero-3-Phosphocholine (DOPC) and two-phase lipid bilayer membranes containing DOPC and 1,2-Distearoyl-Glycero-3-Phosphocholine (DSPC) have been successfully assembled on these surfaces and characterized using fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) to determine the lateral mobility and diffusion coefficients of these unique systems. It was found that xerogel films with rough surface topography lead to slower lipid diffusion due to obstruction of the membrane. Further work is needed to determine if more complexities, which are found in real cell membranes, can be accommodated by these new substrate chemistries. Additionally, sugar-based aerogel and xerogel materials were synthesized for catalytic applications. These unique materials were characterized with SEM and BET surface area and pore size analysis and it was found that sucrose aerogels, with high surface area and smaller pore diameters, are the most promising sugar-based material studied for further catalysis research.

  16. Molecular dynamics simulation of an archaeal lipid bilayer with sodium chloride.

    PubMed

    Shinoda, Keiko; Shinoda, Wataru; Mikami, Masuhiro

    2007-02-01

    We have performed molecular dynamics simulations of a bilayer formed by the synthetic archaeal lipid, diphytanyl phosphatidylcholine, in NaCl electrolyte solution at four different concentrations (0-4 M) to investigate how structural and dynamic properties of the model archaeal membrane are changed due to the ionic strength of the solution. The archaeal lipid bilayer shows minor changes in their physical properties, indicating the unusual high stability of the membrane against salt, though small reductions of molecular area and lateral diffusion of the lipid are detected at the highest electrolyte concentration of 4 M. Sodium ions penetrate to the ether-rich region, where the ions are likely bound to the ether oxygen in the sn-1 chain rather than to that in the sn-2 chain. The observed salt bridges among two or three neighboring lipids account for the small reduction in the molecular area. The bound ions together with the counter (chloride) ions give rise to a diffusive electric double layer; as a result, the membrane dipole potential is slightly increased with increasing NaCl concentration.

  17. Photosensitizer binding to lipid bilayers as a precondition for the photoinactivation of membrane channels.

    PubMed Central

    Rokitskaya, T I; Block, M; Antonenko, Y N; Kotova, E A; Pohl, P

    2000-01-01

    The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcS(n), 1 lipids. Adsorbing to the surface of large unilamellar vesicles (LUVs), aluminum phthalocyanine disulfonate induced the highest changes in their electrophoretic mobility. AlPcS(2) was also most efficient in mediating photoinactivation of gramicidin channels, as revealed by measurements of the electric current across planar lipid bilayers. The increase in the degree of sulfonation of phthalocyanine progressively reduced its affinity for the lipid bilayer as well as its potency of sensitizing gramicidin channel photoinactivation. The portion of photoinactivated gramicidin channels, alpha, increased with rising photosensitizer concentration up to some optimum. The concentration at which alpha was at half-maximum amounted to 80 nM, 30 nM, 200 nM, and 2 microM for AlPcS(1), AlPcS(2), AlPcS(3), and AlPcS(4), respectively. At high concentrations alpha was found to decrease, which was attributed to quenching of reactive oxygen species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both AlPcS(n) (2 lipids is a prerequisite for the photodynamic inactivation of gramicidin channels. PMID:10777753

  18. The effect of tail-length mismatch in binary DMPC/DSPC lipid bilayers

    NASA Astrophysics Data System (ADS)

    Ashkar, Rana; Nagao, Michihiro; Butler, Paul

    2014-03-01

    Bilayer heterogeneity has been long hypothesized to drive raft formation and promote complex functionality in lipid membranes. The highly dynamic nature of the membrane however is thought to play a critical role in this delicate balance between structure and performance. To probe the effect of lateral heterogeneity on membrane dynamics, we investigate the thermal response of unilamellar-vesicle systems of mixed dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) with DMPC/DSPC ratios of 50/50 and 70/30. Both lipids experience a transition from an ordered gel phase, with stiff stretched tails, to a melted fluid phase, with more coiled flexible tails, as they are heated through their melting temperature, Tm(DMPC) ~ 21 °C and Tm(DSPC) ~ 51 °C. The distinct Tm's of the two lipids provide a broad gel-fluid phase with a significant mismatch (~ 20 Å) between the tail-lengths of the DMPC and DSPC molecules. The structural properties of the vesicles were determined by small-angle neutron and x-ray scattering and the collective lipid dynamics in the bilayer were investigated by neutron spin-echo (NSE) spectroscopy on selectively deuterated samples. The NSE results indicate a slowdown of thickness fluctuations in the gel-fluid coexistence phase and an intriguingly strong enhancement in the thickness fluctuation amplitude for T >Tm(DSPC) compared to our previous work on single component vesicles.

  19. Thin-film silica sol-gels doped with ion responsive fluorescent lipid bilayers

    SciTech Connect

    Sasaki, D.Y.; Shea, L.E.; Sinclair, M.B.

    1999-01-12

    A metal ion sensitive, fluorescent lipid-b i layer material (5oA PSIDA/DSPC) was successfully immobilized in a silica matrix using a tetramethoxysilane (TMOS) sol-gel procedure. The sol-gel immobilization method was quantitative in the entrapment of seif-assembled Iipid-bilayers and yielded thin films for facile configuration to optical fiber piatforms. The silica matrix was compatible with the solvent sensitive lipid bilayers and provided physical stabilization as well as biological protection. Immobilization in the silica sol-gel produced an added benefit of improving the bilayer's metal ion sensitivity by up to two orders of magnitude. This enhanced performance was attributed to a preconcentrator effect from the anionic surface of the silica matrix. Thin gels (193 micron thickness) were coupled to a bifurcated fiber optic bundle to produce a metal ion sensor probe. Response times of 10 - 15 minutes to 0.1 M CUCIZ were realized with complete regeneration of the sensor using an ethylenediarninetetraacetic acid (EDTA) solution.

  20. Nanoscale, electric field-driven water bridges in vacuum gaps and lipid bilayers.

    PubMed

    Ho, Ming-Chak; Levine, Zachary A; Vernier, P Thomas

    2013-11-01

    Formation of a water bridge across the lipid bilayer is the first stage of pore formation in molecular dynamic (MD) simulations of electroporation, suggesting that the intrusion of individual water molecules into the membrane interior is the initiation event in a sequence that leads to the formation of a conductive membrane pore. To delineate more clearly the role of water in membrane permeabilization, we conducted extensive MD simulations of water bridge formation, stabilization, and collapse in palmitoyloleoylphosphatidylcholine bilayers and in water-vacuum-water systems, in which two groups of water molecules are separated by a 2.8 nm vacuum gap, a simple analog of a phospholipid bilayer. Certain features, such as the exponential decrease in water bridge initiation time with increased external electric field, are similar in both systems. Other features, such as the relationship between water bridge lifetime and the diameter of the water bridge, are quite different between the two systems. Data such as these contribute to a better and more quantitative understanding of the relative roles of water and lipid in membrane electropore creation and annihilation, facilitating a mechanism-driven development of electroporation protocols. These methods can be extended to more complex, heterogeneous systems that include membrane proteins and intracellular and extracellular membrane attachments, leading to more accurate models of living cells in electric fields. PMID:23644990

  1. Nanoscale, electric field-driven water bridges in vacuum gaps and lipid bilayers.

    PubMed

    Ho, Ming-Chak; Levine, Zachary A; Vernier, P Thomas

    2013-11-01

    Formation of a water bridge across the lipid bilayer is the first stage of pore formation in molecular dynamic (MD) simulations of electroporation, suggesting that the intrusion of individual water molecules into the membrane interior is the initiation event in a sequence that leads to the formation of a conductive membrane pore. To delineate more clearly the role of water in membrane permeabilization, we conducted extensive MD simulations of water bridge formation, stabilization, and collapse in palmitoyloleoylphosphatidylcholine bilayers and in water-vacuum-water systems, in which two groups of water molecules are separated by a 2.8 nm vacuum gap, a simple analog of a phospholipid bilayer. Certain features, such as the exponential decrease in water bridge initiation time with increased external electric field, are similar in both systems. Other features, such as the relationship between water bridge lifetime and the diameter of the water bridge, are quite different between the two systems. Data such as these contribute to a better and more quantitative understanding of the relative roles of water and lipid in membrane electropore creation and annihilation, facilitating a mechanism-driven development of electroporation protocols. These methods can be extended to more complex, heterogeneous systems that include membrane proteins and intracellular and extracellular membrane attachments, leading to more accurate models of living cells in electric fields.

  2. Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity.

    PubMed

    Maher, Sean; Basit, Hajra; Forster, Robert J; Keyes, Tia E

    2016-12-01

    Microcavity supported lipid bilayers, MSLBs, were applied to an electrochemical investigation of ionophore mediated ion transport. The arrays comprise of a 1cm(2) gold electrode imprinted with an ordered array of uniform spherical-cap pores of 2.8μm diameter prepared by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aqueous buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by the micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coefficient of 12.58±1.28μm(2)s(-1) and anomalous exponent of 1.03±0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3days and electrochemical impedance spectroscopy of the membrane with and without ionic gradient over experimental windows of 6h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K(+) uniporter and Nigericin, a K(+)/H(+) antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance respectively. Significant increases in Valinomycin and Nigericin activity were observed, reflected in large decreases in membrane resistance when K(+) was present in the contacting buffer and in the presence of H(+) ionic gradient across the membrane respectively. PMID:27420132

  3. Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity.

    PubMed

    Maher, Sean; Basit, Hajra; Forster, Robert J; Keyes, Tia E

    2016-12-01

    Microcavity supported lipid bilayers, MSLBs, were applied to an electrochemical investigation of ionophore mediated ion transport. The arrays comprise of a 1cm(2) gold electrode imprinted with an ordered array of uniform spherical-cap pores of 2.8μm diameter prepared by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aqueous buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by the micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coefficient of 12.58±1.28μm(2)s(-1) and anomalous exponent of 1.03±0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3days and electrochemical impedance spectroscopy of the membrane with and without ionic gradient over experimental windows of 6h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K(+) uniporter and Nigericin, a K(+)/H(+) antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance respectively. Significant increases in Valinomycin and Nigericin activity were observed, reflected in large decreases in membrane resistance when K(+) was present in the contacting buffer and in the presence of H(+) ionic gradient across the membrane respectively.

  4. Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

    PubMed Central

    Endo, Masayuki; Sugiyama, Hiroshi

    2015-01-01

    Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed ‘lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space. PMID:26310995

  5. Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.

    PubMed

    Schadauer, Florian; Geiss, Andreas F; Srajer, Johannes; Siebenhofer, Bernhard; Frank, Pinar; Reiner-Rozman, Ciril; Ludwig, Bernd; Richter, Oliver-M H; Nowak, Christoph; Naumann, Renate L C

    2015-03-01

    An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.

  6. Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer

    PubMed Central

    Gnanasambandam, Radhakrishnan; Sachs, Frederick

    2016-01-01

    The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (Popen) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, Popen is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases Popen. Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of Popen to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting Popen. The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates. SIGNIFICANCE STATEMENT Classically, changes in extracellular calcium and voltage affect open probability (Popen) through mechanoelectric transduction adaptation, and this mechanism is the only means of controlling the set point of the channel. Here, we further characterize the effects of extracellular calcium and voltage on the channel and for the first time determine that these manipulations occur through a mechanism that is independent of fast adaptation

  7. Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

    PubMed

    Shoji, Atsushi; Ikeya, Kana; Aoyagi, Miki; Takatsuji, Ryutaro; Yanagida, Akio; Shibusawa, Yoichi; Sugawara, Masao

    2016-09-01

    Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the β-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanollipid bilayer. The potential of the SLO nanopore-based method for monitoring cholesterol oxidation in a lipid bilayer by other oxidative enzymes is also discussed.

  8. Formation of lipid bilayers inside microfluidic channel array for monitoring membrane-embedded nanopores of phi29 DNA packaging nanomotor.

    PubMed

    Shim, Joon S; Geng, Jia; Ahn, Chong H; Guo, Peixuan

    2012-10-01

    An efficient method to form lipid bilayers inside an array of microfluidic channels has been developed and applied to monitor the membrane-embedded phi29 DNA packaging motor with an electrochemical characterization on a lab-on-a-chip (LOC) platform. A push-pull junction capturing approach was applied to confine a small amount of the lipid solution inside a microchannel. The selective permeability between solvents and water in PDMS was utilized to extract the solvent from the lipid solution, resulting in a self-formation of the lipid bilayer in the microchannel array. Each microchannel was independently connected to a silver/silver chloride (Ag/AgCl) electrode array, leading to a high-throughput monitoring of the nanopore insertion in the formed lipid bilayers. The formation of multiple lipid bilayers inside an array of microchannels and the simultaneous electrical and optical monitoring of multiple bilayer provides an efficient LOC platform for the further development of single phi29 motor pore sensing and high throughput single pore dsDNA sequencing. PMID:22773160

  9. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    PubMed Central

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  10. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

    PubMed Central

    Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

    2016-01-01

    Local surface charge density of lipid membranes influences membrane–protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values. PMID:27561322

  11. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

    PubMed

    Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

    2016-01-01

    Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values. PMID:27561322

  12. Exploring the Effects on Lipid Bilayer Induced by Noble Gases via Molecular Dynamics Simulations.

    PubMed

    Chen, Junlang; Chen, Liang; Wang, Yu; Wang, Xiaogang; Zeng, Songwei

    2015-01-01

    Noble gases seem to have no significant effect on the anesthetic targets due to their simple, spherical shape. However, xenon has strong narcotic efficacy and can be used clinically, while other noble gases cannot. The mechanism remains unclear. Here, we performed molecular dynamics simulations on phospholipid bilayers with four kinds of noble gases to elucidate the difference of their effects on the membrane. Our results showed that the sequence of effects on membrane exerted by noble gases from weak to strong was Ne, Ar, Kr and Xe, the same order as their relative narcotic potencies as well as their lipid/water partition percentages. Compared with the other three kinds of noble gases, more xenon molecules were distributed between the lipid tails and headgroups, resulting in membrane's lateral expansion and lipid tail disorder. It may contribute to xenon's strong anesthetic potency. The results are well consistent with the membrane mediated mechanism of general anesthesia. PMID:26601882

  13. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

    NASA Astrophysics Data System (ADS)

    Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

    2016-08-01

    Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

  14. Exploring the Effects on Lipid Bilayer Induced by Noble Gases via Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Chen, Junlang; Chen, Liang; Wang, Yu; Wang, Xiaogang; Zeng, Songwei

    2015-11-01

    Noble gases seem to have no significant effect on the anesthetic targets due to their simple, spherical shape. However, xenon has strong narcotic efficacy and can be used clinically, while other noble gases cannot. The mechanism remains unclear. Here, we performed molecular dynamics simulations on phospholipid bilayers with four kinds of noble gases to elucidate the difference of their effects on the membrane. Our results showed that the sequence of effects on membrane exerted by noble gases from weak to strong was Ne, Ar, Kr and Xe, the same order as their relative narcotic potencies as well as their lipid/water partition percentages. Compared with the other three kinds of noble gases, more xenon molecules were distributed between the lipid tails and headgroups, resulting in membrane’s lateral expansion and lipid tail disorder. It may contribute to xenon’s strong anesthetic potency. The results are well consistent with the membrane mediated mechanism of general anesthesia.

  15. Theory of Kinetics of Registration and Anti-Registration in Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Olmsted, Peter; Williamson, John

    Lipid bilayer leaflets are often treated as if they are coupled; i.e., that the two leaflets undergo simultaneous transitions between phases, and that domains involve both leaflets together in a registered fashion. We present theory and simulation showing how interleaflet couplings and hydrophobic mismatch can lead to a complex phase diagram with multiple metastable two-phase and three-phase states. Many of these states can be discerned in the experimental literature, and are expected in the early stages of coarsening when domains are sub-micron (and thus perhaps of significance to lipid rafts). We present different kinetic scenarios for transitions between these state, and show how lipid flip flop can surprisingly lead to non-symmetric anti-registered patterns.

  16. Voltage-controlled insertion of single α-hemolysin and Mycobacterium smegmatis nanopores into lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Renner, Stephan; Bessonov, Andrey; Simmel, Friedrich C.

    2011-02-01

    One of the prerequisites for single molecule nanopore translocation experiments is the availability of a single nanopore embedded into a lipid bilayer membrane. Using two alternative experimental setups, microdroplets, and a classical planar lipid bilayer setup, we here show that at near-neutral pH and high salt concentration the incorporation rates of the pore-forming proteins α-hemolysin and porin A from Mycobacterium smegmatis are exponentially enhanced at elevated voltages. This fact can be utilized to establish an experimental procedure by which a voltage-controlled insertion of single pores into lipid membranes can be achieved.

  17. A Distributed Amplifier System for Bilayer Lipid Membrane (BLM) Arrays With Noise and Individual Offset Cancellation.

    PubMed

    Crescentini, Marco; Thei, Frederico; Bennati, Marco; Saha, Shimul; de Planque, Maurits R R; Morgan, Hywel; Tartagni, Marco

    2015-06-01

    Lipid bilayer membrane (BLM) arrays are required for high throughput analysis, for example drug screening or advanced DNA sequencing. Complex microfluidic devices are being developed but these are restricted in terms of array size and structure or have integrated electronic sensing with limited noise performance. We present a compact and scalable multichannel electrophysiology platform based on a hybrid approach that combines integrated state-of-the-art microelectronics with low-cost disposable fluidics providing a platform for high-quality parallel single ion channel recording. Specifically, we have developed a new integrated circuit amplifier based on a novel noise cancellation scheme that eliminates flicker noise derived from devices under test and amplifiers. The system is demonstrated through the simultaneous recording of ion channel activity from eight bilayer membranes. The platform is scalable and could be extended to much larger array sizes, limited only by electronic data decimation and communication capabilities.

  18. Nanoscale measurement of the dielectric constant of supported lipid bilayers in aqueous solutions with electrostatic force microscopy.

    PubMed

    Gramse, G; Dols-Perez, A; Edwards, M A; Fumagalli, L; Gomila, G

    2013-03-19

    We present what is, to our knowledge, the first experimental demonstration of dielectric constant measurement and quantification of supported lipid bilayers in electrolyte solutions with nanoscale spatial resolution. The dielectric constant was quantitatively reconstructed with finite element calculations by combining thickness information and local polarization forces which were measured using an electrostatic force microscope adapted to work in a liquid environment. Measurements of submicrometric dipalmitoylphosphatidylcholine lipid bilayer patches gave dielectric constants of ε(r) ~ 3, which are higher than the values typically reported for the hydrophobic part of lipid membranes (ε(r) ~ 2) and suggest a large contribution of the polar headgroup region to the dielectric response of the lipid bilayer. This work opens apparently new possibilities in the study of biomembrane electrostatics and other bioelectric phenomena.

  19. Squalane is in the midplane of the lipid bilayer: implications for its function as a proton permeability barrier.

    PubMed

    Hauss, Thomas; Dante, Silvia; Dencher, Norbert A; Haines, Thomas H

    2002-12-01

    A recently proposed model for proton leakage across biological membranes [Prog. Lipid Res. 40 (2001) 299] suggested that hydrocarbons specifically in the center of the lipid bilayer inhibit proton leaks. Since cellular membranes maintain a proton electrochemical gradient as a principal energy transducer, proton leakage unproductively consumes cellular energy. Hydrocarbons in the bilayer are widespread in membranes that sustain such gradients. The alkaliphiles are unique in that they contain up to 40 mol% isoprenes in their membranes including 10-11 mol% squalene [J. Bacteriol. 168 (1986) 334]. Squalene is a polyisoprene hydrocarbon without polar groups. Localizing hydrocarbons in lipid bilayers has not been trivial. A myriad of physical methods including fluorescence spectroscopy, electron-spin resonance, nuclear magnetic resonance as well as X-ray and neutron diffraction have been used to explore this question with various degrees of success and often contradictory results. Seeking unambiguous evidence for the localization of squalene in membranes or lipid bilayers, we employed neutron diffraction. We incorporated 10 mol% perdeuterated or protonated squalane, an isosteric analogue of squalene, into stacked bilayers of dioleoyl phosphatidyl choline (DOPC) doped with dioleoyl phosphatidyl glycerol (DOPG) to simulate the negative charges found on natural membranes. The neutron diffraction data clearly show that the squalane lies predominantly in the bilayer center, parallel to the plane of the membrane.

  20. Data supporting beta-amyloid dimer structural transitions and protein–lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures

    PubMed Central

    Cheng, Sara Y.; Chou, George; Buie, Creighton; Vaughn, Mark W.; Compton, Campbell; Cheng, Kwan H.

    2016-01-01

    This data article supports the research article entitled “Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface” [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes. PMID:27054174

  1. Data supporting beta-amyloid dimer structural transitions and protein-lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures.

    PubMed

    Cheng, Sara Y; Chou, George; Buie, Creighton; Vaughn, Mark W; Compton, Campbell; Cheng, Kwan H

    2016-06-01

    This data article supports the research article entitled "Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface" [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes.

  2. Chemotherapy Drugs Thiocolchicoside and Taxol Permeabilize Lipid Bilayer Membranes by Forming Ion Pores

    NASA Astrophysics Data System (ADS)

    Ashrafuzzaman, Md; Duszyk, M.; Tuszynski, J. A.

    2011-12-01

    We report ion channel formation by chemotherapy drugs: thiocolchicoside (TCC) and taxol (TXL) which primarily target tubulin but not only. For example, TCC has been shown to interact with GABAA, nuclear envelope and strychnine-sensitive glycine receptors. TXL interferes with the normal breakdown of microtubules inducing mitotic block and apoptosis. It also interacts with mitochondria and found significant chemotherapeutic applications for breast, ovarian and lung cancer. In order to better understand the mechanisms of TCC and TXL actions, we examined their effects on phospholipid bilayer membranes. Our electrophysiological recordings across membranes constructed in NaCl aqueous phases consisting of TCC or TXL under the influence of an applied transmembrane potential (V) indicate that both molecules induce stable ion flowing pores/channels in membranes. Their discrete current versus time plots exhibit triangular shapes which is consistent with a spontaneous time-dependent change of the pore conductance in contrast to rectangular conductance events usually induced by ion channels. These events exhibit conductance (~0.01-0.1 pA/mV) and lifetimes (~5-30 ms) within the ranges observed in e.g., gramicidin A and alamethicin channels. The channel formation probability increases linearly with TCC/TXL concentration and V and is not affected by pH (5.7 - 8.4). A theoretical explanation on the causes of chemotherapy drug induced ion pore formation and the pore stability has also been found using our recently discovered binding energy between lipid bilayer and the bilayer embedded ion channels using gramicidin A channels as tools. This picture of energetics suggests that as the channel forming agents approach to the lipids on bilayer the localized charge properties in the constituents of both channel forming agents (e.g., chemotherapy drugs in this study) and the lipids determine the electrostatic drug-lipid coupling energy through screened Coulomb interactions between the drug

  3. Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer.

    PubMed

    McIlwain, Benjamin C; Vandenberg, Robert J; Ryan, Renae M

    2015-04-10

    The aspartate transporter from Pyrococcus horikoshii (GltPh) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of GltPh provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting GltPh into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of GltPh were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of GltPh revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.

  4. The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

    PubMed

    Chadda, Rahul; Krishnamani, Venkatramanan; Mersch, Kacey; Wong, Jason; Brimberry, Marley; Chadda, Ankita; Kolmakova-Partensky, Ludmila; Friedman, Larry J; Gelles, Jeff; Robertson, Janice L

    2016-01-01

    Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

  5. Interactions of Graphene Oxide with Model Cell Membranes: Probing Nanoparticle Attachment and Lipid Bilayer Disruption.

    PubMed

    Liu, Xitong; Chen, Kai Loon

    2015-11-10

    With the rapid growth in the application of graphene oxide (GO) in diverse fields, the toxicity of GO toward bacterial and mammalian cells has recently attracted extensive research attention. While several mechanisms have been proposed for the cytotoxicity of GO, the attachment of GO to cell membranes is expected to be the key initial process that precedes these mechanisms. In this study, we investigate the propensity for GO to attach to and disrupt model cell membranes using supported lipid bilayers (SLBs) and supported vesicular layers (SVLs) that are composed of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The deposition kinetics of GO on SLBs were determined using quartz crystal microbalance with dissipation monitoring and were observed to increase with increasing electrolyte (NaCl and CaCl2) concentrations, indicating that GO attachment to SLBs was controlled by electrostatic interactions. The GO deposition kinetics measured at elevated electrolyte concentrations were lower than mass-transfer-limited kinetics, likely due to the presence of hydration forces between GO and SLBs. Upon the attachment of GO to supported vesicles that were encapsulated with a fluorescent dye, dye leakage was detected, thus indicating that the lipid vesicles were disrupted. When the exposure of the SVL to the GO suspension was terminated, the leakage of dye decreased significantly, demonstrating that the pores on the lipid bilayers have a self-healing ability. PMID:26466194

  6. The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers

    PubMed Central

    Chadda, Rahul; Krishnamani, Venkatramanan; Mersch, Kacey; Wong, Jason; Brimberry, Marley; Chadda, Ankita; Kolmakova-Partensky, Ludmila; Friedman, Larry J; Gelles, Jeff; Robertson, Janice L

    2016-01-01

    Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes. DOI: http://dx.doi.org/10.7554/eLife.17438.001 PMID:27484630

  7. Nanoarrays of tethered lipid bilayer rafts on poly(vinyl alcohol) hydrogels.

    PubMed

    Lee, Bong Kuk; Lee, Hea Yeon; Kim, Pilnam; Suh, Kahp Y; Kawai, Tomoji

    2009-01-01

    Lipid rafts are cholesterol- and sphingolipid-rich domains that function as platforms for signal transduction and other cellular processes. Tethered lipid bilayers have been proposed as a promising model to describe the structure and function of cell membranes. We report a nano(submicro) array of tethered lipid bilayer raft membranes (tLBRMs) comprising a biosensing platform. Poly(vinyl alcohol) (PVA) hydrogel was directly patterned onto a solid substrate, using ultraviolet-nanoimprint lithography (UV-NIL), as an inert barrier to prevent biofouling. The robust structures of the nanopatterned PVA hydrogel were stable for up to three weeks in phosphate-buffered saline solution despite significant swelling (100% in height) by hydration. The PVA hydrogel strongly restricted the adhesion of vesicles, resulting in an array of highly selective hydrogel nanowells. tLBRMs were not formed by direct vesicle fusion, although raft vesicles containing poly(ethylene glycol) lipopolymer were selectively immobilized on gold substrates patterned with PVA hydrogel. The deposition of tLBRM nano(submicro) arrays was accomplished by a mixed, self-assembled monolayer-assisted vesicle fusion method. The monolayer was composed of a mixture of 2-mercaptoethanol and poly(ethylene glycol) lipopolymer, which promoted vesicle rupture. These results suggest that the fabrication of inert nanostructures and the site-selective modification of solid surfaces to induce vesicle rupture may be essential in the construction of tLBRM nano(submicro) arrays using stepwise self-assembly.

  8. Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes.

    PubMed

    Geng, Jia; Kim, Kyunghoon; Zhang, Jianfei; Escalada, Artur; Tunuguntla, Ramya; Comolli, Luis R; Allen, Frances I; Shnyrova, Anna V; Cho, Kang Rae; Munoz, Dayannara; Wang, Y Morris; Grigoropoulos, Costas P; Ajo-Franklin, Caroline M; Frolov, Vadim A; Noy, Aleksandr

    2014-10-30

    There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100 picosiemens under physiological conditions. Despite their structural simplicity, these 'CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

  9. Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes

    NASA Astrophysics Data System (ADS)

    Geng, Jia; Kim, Kyunghoon; Zhang, Jianfei; Escalada, Artur; Tunuguntla, Ramya; Comolli, Luis R.; Allen, Frances I.; Shnyrova, Anna V.; Cho, Kang Rae; Munoz, Dayannara; Wang, Y. Morris; Grigoropoulos, Costas P.; Ajo-Franklin, Caroline M.; Frolov, Vadim A.; Noy, Aleksandr

    2014-10-01

    There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100 picosiemens under physiological conditions. Despite their structural simplicity, these `CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

  10. Preparation of mica supported lipid bilayers for high resolution optical microscopy imaging.

    PubMed

    Matysik, Artur; Kraut, Rachel S

    2014-06-07

    Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

  11. Molecular dynamics in lipid bilayers. Anisotropic diffusion in an odd restoring potential.

    PubMed Central

    Alam, T M

    1993-01-01

    Recent 2H nuclear magnetic resonance spin relaxation studies have questioned the influence of restoring potential parity on the description of lipid or molecular reorientational dynamics. In biomembranes the polar head groups of lipid and sterol constituents are expected to associate with the aqueous interface; therefore, realistic descriptions of molecular reorientation in bilayer systems should use an odd restoring potential. The multiexponential correlation functions and related spectral density functions for small-step anisotropic diffusion in a pseudo-restoring potential of the form U(beta) = -lambda cos beta are evaluated as a function of molecular ordering . From analysis of these results the single exponential approximation used in previous investigations is found to overestimate the decay rate at lower order, but is reliable for > 0.6. The exception to this trend is the decay constant for the G11 (omega; t) correlation function, which is not accurately portrayed by the first-order approximations. A second-order single exponential approximation is presented, and is shown to be equivalent to the weighted sum of the multidecay constants. In general, the potential parity is found to have only minor effects on the spin relaxation rates obtained to describe molecular reorientation in lipid bilayers. PMID:8369401

  12. Solution-Based Single-Molecule FRET Studies of K(+) Channel Gating in a Lipid Bilayer.

    PubMed

    Sadler, Emma E; Kapanidis, Achillefs N; Tucker, Stephen J

    2016-06-21

    Ion channels are dynamic multimeric proteins that often undergo multiple unsynchronized structural movements as they switch between their open and closed states. Such structural changes are difficult to measure within the context of a native lipid bilayer and have often been monitored via macroscopic changes in Förster resonance energy transfer (FRET) between probes attached to different parts of the protein. However, the resolution of this approach is limited by ensemble averaging of structurally heterogeneous subpopulations. These problems can be overcome by measurement of FRET in single molecules, but this presents many challenges, in particular the ability to control labeling of subunits within a multimeric protein with acceptor and donor fluorophores, as well as the requirement to image large numbers of individual molecules in a membrane environment. To address these challenges, we randomly labeled tetrameric KirBac1.1 potassium channels, reconstituted them into lipid nanodiscs, and performed single-molecule FRET confocal microscopy with alternating-laser excitation as the channels diffused in solution. These solution-based single-molecule FRET measurements of a multimeric ion channel in a lipid bilayer have allowed us to probe the structural changes that occur upon channel activation and inhibition. Our results provide direct evidence of the twist-to-shrink movement of the helix bundle crossing during channel gating and demonstrate how this method might be applied to real-time structural studies of ion channel gating. PMID:27332124

  13. Kinetic Control of Histidine-Tagged Protein Surface Density on Supported Lipid Bilayers

    SciTech Connect

    Nye, Jeffrey A.; Groves, Jay T.

    2008-02-28

    Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. In this study, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 ≈ 6 min), whereas polyvalently bound species remain bound for hours (t1/2 ≈ 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. In conclusion, we propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.

  14. Asymmetric distribution of charged lipids between the leaflets of a vesicle bilayer induced by melittin and alamethicin

    SciTech Connect

    Qian, Shuo; Heller, William T

    2011-01-01

    Cellular membranes are complex mixtures of lipids, proteins, and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. The membrane structure results from the complex interplay between the wide varieties of molecules present. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoyl phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than it is for alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state. The results suggest that these membrane-active peptides may have a secondary stressful effect on target cells at low concentrations that results from a disruption of the lipid distribution between the inner and outer leaflets of the bilayer that is independent of the formation of transmembrane pores.

  15. Atomistic resolution structure and dynamics of lipid bilayers in simulations and experiments.

    PubMed

    Ollila, O H Samuli; Pabst, Georg

    2016-10-01

    Accurate details on the sampled atomistic resolution structures of lipid bilayers can be experimentally obtained by measuring C-H bond order parameters, spin relaxation rates and scattering form factors. These parameters can be also directly calculated from the classical atomistic resolution molecular dynamics simulations (MD) and compared to the experimentally achieved results. This comparison measures the simulation model quality with respect to 'reality'. If agreement is sufficient, the simulation model gives an atomistic structural interpretation of the acquired experimental data. Significant advance of MD models is made by jointly interpreting different experiments using the same structural model. Here we focus on phosphatidylcholine lipid bilayers, which out of all model membranes have been studied mostly by experiments and simulations, leading to the largest available dataset. From the applied comparisons we conclude that the acyl chain region structure and rotational dynamics are generally well described in simulation models. Also changes with temperature, dehydration and cholesterol concentration are qualitatively correctly reproduced. However, the quality of the underlying atomistic resolution structural changes is uncertain. Even worse, when focusing on the lipid bilayer properties at the interfacial region, e.g. glycerol backbone and choline structures, and cation binding, many simulation models produce an inaccurate description of experimental data. Thus extreme care must be applied when simulations are applied to understand phenomena where the interfacial region plays a significant role. This work is done by the NMRlipids Open Collaboration project running at https://nmrlipids.blogspot.fi and https://github.com/NMRLipids. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

  16. Improved Experimental Techniques for Analyzing Nucleic Acid Transport Through Protein Nanopores in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Costa, Justin A.

    The translocation of nucleic acid polymers across cell membranes is a fundamental requirement for complex life and has greatly contributed to genomic molecular evolution. The diversity of pathways that have evolved to transport DNA and RNA across membranes include protein receptors, active and passive transporters, endocytic and pinocytic processes, and various types of nucleic acid conducting channels known as nanopores. We have developed a series of experimental techniques, collectively known as "Wicking", that greatly improves the biophysical analysis of nucleic acid transport through protein nanopores in planar lipid bilayers. We have verified the Wicking method using numerous types of classical ion channels including the well-studied chloride selective channel, CLIC1. We used the Wicking technique to reconstitute α-hemolysin and found that DNA translocation events of types A and B could be routinely observed using this method. Furthermore, measurable differences were observed in the duration of blockade events as DNA length and composition was varied, consistent with previous reports. Finally, we tested the ability of the Wicking technology to reconstitute the dsRNA transporter Sid-1. Exposure to dsRNAs of increasing length and complexity showed measurable differences in the current transitions suggesting that the charge carrier was dsRNA. However, the translocation events occurred so infrequently that a meaningful electrophysiological analysis was not possible. Alterations in the lipid composition of the bilayer had a minor effect on the frequency of translocation events but not to such a degree as to permit rigorous statistical analysis. We conclude that in many instances the Wicking method is a significant improvement to the lipid bilayer technique, but is not an optimal method for analyzing transport through Sid-1. Further refinements to the Wicking method might have future applications in high throughput DNA sequencing, DNA computation, and

  17. Permeation of the three aromatic dipeptides through lipid bilayers: Experimental and computational study.

    PubMed

    Lee, Brent L; Kuczera, Krzysztof; Middaugh, C Russell; Jas, Gouri S

    2016-06-28

    The time-resolved parallel artificial membrane permeability assay with fluorescence detection and comprehensive computer simulations are used to study the passive permeation of three aromatic dipeptides-N-acetyl-phenylalanineamide (NAFA), N-acetyltyrosineamide (NAYA), and N-acetyl-tryptophanamide (NATA) through a 1,2-dioleoyl-sn-glycero-3-phospocholine (DOPC) lipid bilayer. Measured permeation times and permeability coefficients show fastest translocation for NAFA, slowest for NAYA, and intermediate for NATA under physiological temperature and pH. Computationally, we perform umbrella sampling simulations to model the structure, dynamics, and interactions of the peptides as a function of z, the distance from lipid bilayer. The calculated profiles of the potential of mean force show two strong effects-preferential binding of each of the three peptides to the lipid interface and large free energy barriers in the membrane center. We use several approaches to calculate the position-dependent translational diffusion coefficients D(z), including one based on numerical solution the Smoluchowski equation. Surprisingly, computed D(z) values change very little with reaction coordinate and are also quite similar for the three peptides studied. In contrast, calculated values of sidechain rotational correlation times τrot(z) show extremely large changes with peptide membrane insertion-values become 100 times larger in the headgroup region and 10 times larger at interface and in membrane center, relative to solution. The peptides' conformational freedom becomes systematically more restricted as they enter the membrane, sampling α and β and C7eq basins in solution, α and C7eq at the interface, and C7eq only in the center. Residual waters of solvation remain around the peptides even in the membrane center. Overall, our study provides an improved microscopic understanding of passive peptide permeation through membranes, especially on the sensitivity of rotational diffusion to

  18. Permeation of the three aromatic dipeptides through lipid bilayers: Experimental and computational study

    NASA Astrophysics Data System (ADS)

    Lee, Brent L.; Kuczera, Krzysztof; Middaugh, C. Russell; Jas, Gouri S.

    2016-06-01

    The time-resolved parallel artificial membrane permeability assay with fluorescence detection and comprehensive computer simulations are used to study the passive permeation of three aromatic dipeptides - N-acetyl-phenylalanineamide (NAFA), N-acetyltyrosineamide (NAYA), and N-acetyl-tryptophanamide (NATA) through a 1,2-dioleoyl-sn-glycero-3-phospocholine (DOPC) lipid bilayer. Measured permeation times and permeability coefficients show fastest translocation for NAFA, slowest for NAYA, and intermediate for NATA under physiological temperature and pH. Computationally, we perform umbrella sampling simulations to model the structure, dynamics, and interactions of the peptides as a function of z, the distance from lipid bilayer. The calculated profiles of the potential of mean force show two strong effects - preferential binding of each of the three peptides to the lipid interface and large free energy barriers in the membrane center. We use several approaches to calculate the position-dependent translational diffusion coefficients D(z), including one based on numerical solution the Smoluchowski equation. Surprisingly, computed D(z) values change very little with reaction coordinate and are also quite similar for the three peptides studied. In contrast, calculated values of sidechain rotational correlation times τrot(z) show extremely large changes with peptide membrane insertion - values become 100 times larger in the headgroup region and 10 times larger at interface and in membrane center, relative to solution. The peptides' conformational freedom becomes systematically more restricted as they enter the membrane, sampling α and β and C7eq basins in solution, α and C7eq at the interface, and C7eq only in the center. Residual waters of solvation remain around the peptides even in the membrane center. Overall, our study provides an improved microscopic understanding of passive peptide permeation through membranes, especially on the sensitivity of rotational diffusion

  19. Atomistic resolution structure and dynamics of lipid bilayers in simulations and experiments.

    PubMed

    Ollila, O H Samuli; Pabst, Georg

    2016-10-01

    Accurate details on the sampled atomistic resolution structures of lipid bilayers can be experimentally obtained by measuring C-H bond order parameters, spin relaxation rates and scattering form factors. These parameters can be also directly calculated from the classical atomistic resolution molecular dynamics simulations (MD) and compared to the experimentally achieved results. This comparison measures the simulation model quality with respect to 'reality'. If agreement is sufficient, the simulation model gives an atomistic structural interpretation of the acquired experimental data. Significant advance of MD models is made by jointly interpreting different experiments using the same structural model. Here we focus on phosphatidylcholine lipid bilayers, which out of all model membranes have been studied mostly by experiments and simulations, leading to the largest available dataset. From the applied comparisons we conclude that the acyl chain region structure and rotational dynamics are generally well described in simulation models. Also changes with temperature, dehydration and cholesterol concentration are qualitatively correctly reproduced. However, the quality of the underlying atomistic resolution structural changes is uncertain. Even worse, when focusing on the lipid bilayer properties at the interfacial region, e.g. glycerol backbone and choline structures, and cation binding, many simulation models produce an inaccurate description of experimental data. Thus extreme care must be applied when simulations are applied to understand phenomena where the interfacial region plays a significant role. This work is done by the NMRlipids Open Collaboration project running at https://nmrlipids.blogspot.fi and https://github.com/NMRLipids. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26809025

  20. Physical encapsulation and controlled assembly of lipid bilayers within flexible substrates

    NASA Astrophysics Data System (ADS)

    Sarles, Stephen A.; Leo, Donald J.

    2010-04-01

    Biomolecular networks formed from droplet interface bilayers (DIB) use principles of phase separation and molecular self-assembly to create a new type of functional material. The original DIB embodiment consists of lipid-encased aqueous droplets surrounding by a large volume of oil contained in a shallow well. However, recent results have shown that, by reducing the amount of oil that separates the droplets from the supporting substrate, physically-encapsulated DIBs display increased durability and portability. In this paper we extend the concept of encapsulated biomolecular networks to one in which phase separation and molecular self-assembly occur entirely within internally-structured reservoirs of a solid material. Flexible substrates with 200μm wideby- 200μm deep internal microchannels for holding the aqueous and oil phases are fabricated from Sylgard 184 polydimethylsiloxane (PDMS) using soft-lithography microfabrication techniques. Narrowed apertures along the microchannels enable the use of the regulated attachment method (RAM) to subdivide and reattach lipid-encased aqueous volumes contained within the material with an applied external force. The use of perfluorodecalin, a fluorocarbon oil, instead of hexadecane eliminates absorption of the oil phase into the PDMS bulk while a silanization surface treatment of the internal channel walls maximizes wetting by the oil phase to retain a thin layer of oil within the channels to provide a fluid oil/water interface around the aqueous volumes. High-quality 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPHPC) lipid bilayers formed within the prototype substrates have electrical resistance between 1-100GΩ, enabling the measurement of single and few-channel recordings of alpha-hemolysin (αHL) and alamethicin proteins incorporated into the bilayers.

  1. The intermediate scattering function for lipid bilayer membranes: From nanometers to microns

    NASA Astrophysics Data System (ADS)

    Watson, Max C.; Peng, Yonggang; Zheng, Yujun; Brown, Frank L. H.

    2011-11-01

    A numerical scheme based upon established hydrodynamic and elastic considerations is introduced and used to predict the intermediate scattering function for lipid bilayer membranes. The predictions span multiple wavelength regimes, including those studied by dynamic light scattering (DLS; microns) and neutron spin-echo (NSE) spectroscopy (10-100 nm). The results validate a recent theory specific to the NSE regime and expose slight inaccuracies associated with the theoretical results available in the DLS regime. The assumptions that underlie both our numerical methods and the related theoretical predictions are reviewed in detail to explain when certain results can be applied to experiment and where caution must be exercised.

  2. A chloride-permeable channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers.

    PubMed

    Balleza, Daniel; Quinto, Carmen; Sánchez, Federico; Gómez-Lagunas, Froylan

    2003-07-18

    Ion channels are key participants in physiological processes of plant cells. Here, we report the first characterization of a high conductance, Cl(-)-permeable channel, present in enriched fractions of plasma membranes of bean root cells. The Cl(-) channel was incorporated into planar lipid bilayers and its activity was recorded under voltage clamp conditions. The channel is voltage-dependent, excludes the passage of cations (K(+), Na(+), and Ca(2+)), and is inhibited by micromolar concentrations of Zn(2+). The Cl(-) conductance here characterized represents a previously undescribed channel of plant cells.

  3. Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters

    PubMed Central

    Soga, Naoki; Watanabe, Rikiya; Noji, Hiroyuki

    2015-01-01

    We present an attolitre-sized arrayed lipid bilayer chamber system (aL-ALBiC) for rapid and massively parallel single-molecule assay of membrane transporter activity. Because of the small reaction volume (200 aL), the aL-ALBiC performed fast detection of single transporter activity, thereby enhancing the sensitivity, throughput, and accuracy of the analysis. Thus, aL-ALBiC broadens the opportunities for single-molecule analysis of various membrane transporters and can be used in pharmaceutical applications such as drug screening. PMID:26052065

  4. Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters.

    PubMed

    Soga, Naoki; Watanabe, Rikiya; Noji, Hiroyuki

    2015-06-08

    We present an attolitre-sized arrayed lipid bilayer chamber system (aL-ALBiC) for rapid and massively parallel single-molecule assay of membrane transporter activity. Because of the small reaction volume (200 aL), the aL-ALBiC performed fast detection of single transporter activity, thereby enhancing the sensitivity, throughput, and accuracy of the analysis. Thus, aL-ALBiC broadens the opportunities for single-molecule analysis of various membrane transporters and can be used in pharmaceutical applications such as drug screening.

  5. The intermediate scattering function for lipid bilayer membranes: From nanometers to microns

    SciTech Connect

    Watson, Max C.; Peng Yonggang; Zheng Yujun; Brown, Frank L. H.

    2011-11-21

    A numerical scheme based upon established hydrodynamic and elastic considerations is introduced and used to predict the intermediate scattering function for lipid bilayer membranes. The predictions span multiple wavelength regimes, including those studied by dynamic light scattering (DLS; microns) and neutron spin-echo (NSE) spectroscopy (10-100 nm). The results validate a recent theory specific to the NSE regime and expose slight inaccuracies associated with the theoretical results available in the DLS regime. The assumptions that underlie both our numerical methods and the related theoretical predictions are reviewed in detail to explain when certain results can be applied to experiment and where caution must be exercised.

  6. Improved stability of free-standing lipid bilayers based on nanoporous alumina films

    NASA Astrophysics Data System (ADS)

    Hirano-Iwata, Ayumi; Taira, Tasuku; Oshima, Azusa; Kimura, Yasuo; Niwano, Michio

    2010-05-01

    In this study, we propose a method for improving the stability of free-standing bilayer lipid membranes (BLMs) by preparing BLMs across nanoporous alumina films. The use of porous alumina reduced individual membrane size to improve the BLM stability. The BLMs were stable under an applied voltage of ±1 V. The lifetime of the BLMs was 16-30 h with and without incorporated gramicidin channels. Electric properties of the BLMs as a platform for channel-current recordings are also discussed. Since the total area of the BLMs is still large to facilitate protein incorporation, our approach is useful for designing highly sensitive biosensors based on channel proteins.

  7. Structural properties of archaeal lipid bilayers: small-angle X-ray scattering and molecular dynamics simulation study.

    PubMed

    Polak, Andraž; Tarek, Mounir; Tomšič, Matija; Valant, Janez; Ulrih, Nataša Poklar; Jamnik, Andrej; Kramar, Peter; Miklavčič, Damijan

    2014-07-22

    Aeropyrum pernix is an aerobic hyperthermophilic archaeon that grows in harsh environmental conditions and as such possesses unique structural and metabolic features. Its membrane interfaces with the extreme environment and is the first line of defense from external factors. Therefore, lipids composing this membrane have special moieties that increase its stability. The membrane of A. pernix is composed predominantly of two polar lipids 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI). Both have methyl branches in their lipid tails and ether linkages and carbohydrates in their headgroup. These moieties significantly affect the structure and dynamics of the bilayer. To provide a molecular level insight into these characteristics, we used here Molecular Dynamics (MD) simulations of lipid bilayers of composition similar to those of the archaeal membranes. First, we show that the electron density profiles along the normal to the bilayers derived from the simulations are in good agreement with the profiles obtained by the small-angle X-ray scattering (SAXS) technique, which provides confidence in the force fields used. Analyses of the simulation data show that the archaeal lipid bilayers are less hydrated than conventional phosphatidylcholine (PC) lipids and that their structure is not affected by the salt present in the surrounding solution. Furthermore, the lateral pressure in their hydrophobic core, due to the presence of the branched tails, is much higher than that at PC-based lipid bilayers. Both the methyl branched tails and the special headgroup moieties contribute to slow drastically the lateral diffusion of the lipids. Furthermore, we found that the lipid head groups associate via hydrogen bonding, which affects their reorientational dynamics. All together, our data provide links between the microscopic properties of these membranes and their overall

  8. Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

    PubMed

    Jordi, W; Nibbeling, R; de Kruijff, B

    1990-02-12

    The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.

  9. Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.

    PubMed Central

    Ismailov, I I; Berdiev, B K; Shlyonsky, V G; Benos, D J

    1997-01-01

    A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+. Images FIGURE 3 FIGURE 4 PMID:9138565

  10. A Monte Carlo study of peptide insertion into lipid bilayers: equilibrium conformations and insertion mechanisms.

    PubMed Central

    Maddox, Michael W; Longo, Marjorie L

    2002-01-01

    The membrane insertion behavior of two peptides, Magainin2 and M2 delta, was investigated by applying the Monte Carlo simulation technique to a theoretical model. The model included many novel aspects, such as a new semi-empirical lipid bilayer model and a new set of semi-empirical transfer energies, which reproduced the experimental insertion behavior of Magainin2 and M2 delta without parameter fitting. Additionally, we have taken into account diminished internal (intramolecular) hydrogen bonding at the N- and C-termini of helical peptides. All simulations were carried out at 305 K, above the membrane thermal phase transition temperature, and at pH 7.0. The peptide equilibrium conformations are discussed for a range of bilayers with tail polarities varying from octanol-like to alkane-like. Probability distributions of the individual amino-acid-residue positions show the dynamic nature of these equilibrium conformations. Two different insertion mechanisms for M2 delta, and a translocation mechanism for Magainin2, are described. A study of the effect of bilayer thickness on M2 delta insertion suggests a critical thickness above which insertion is unfavorable. Additionally, we did not need to use an orientational potential or array of hard cylinders to persuade M2 delta to insert perpendicular to the membrane surface. Instead, we found that diminished internal hydrogen bonding in the helical conformation anchored the termini in the headgroups and resulted in a nearly perpendicular orientation. PMID:11751313

  11. Solid-state /sup 15/N NMR of oriented lipid bilayer bound gramicidin A'

    SciTech Connect

    Nicholson, L.K.; Moll, F.; Mixon, T.E.; LoGrasso, P.V.; Lay, J.C.; Cross, T.A.

    1987-10-20

    Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28/sup 0/C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +-3/sup 0/. Uniformly /sup 15/N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing /sup 15/N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution /sup 15/N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. The bond orientations are only partially consistent with the extant structural models of gramicidin.

  12. [A study of the inhomogeneity of bilayer lipid membranes by measurements of higher harmonics of transmembrane current].

    PubMed

    Anosov, A A; Barabanenkov, Iu N; Sel'skiĭ, A G

    2006-01-01

    Higher harmonics of alternating current in bilayer lipid membranes caused by sinusoidal voltage applied to the membrane were measured. The bilayer lipid membranes were prepared from diphytanoylphosphatidylcholine in n-decane and n-tetradecane, and measurements were conducted with the aid of an analog-to-digital converter of 16th category. Sinusoidal voltage was formed using a digital-to-analog converter of the 16th category. The dynamic region of measurements was up 90 dB. The results of measurements were used to determine the alpha and beta coefficients of the expansion of membrane capacity C in terms of membrane voltage U C = C0 (1 + alphaU2 + betaU4). We showed in the framework of the electrostriction model that the relation between the alpha and beta coefficients characterizes the inhomogeneity of bilayer lipid membrane with respect to its thickness and Young modulus of elasticity.

  13. PTEN interaction with tethered bilayer lipid membranes containing PI(4,5)P2

    NASA Astrophysics Data System (ADS)

    Moldovan, R.; Shenoy, S.; Shekhar, P.; Kalinowski, A.; Gericke, A.; Heinrich, F.; Loesche, M.

    2009-03-01

    Synthetic lipid membrane models are frequently used for the study of biophysical processes at cell membranes. We use a robust membrane model, the tethered bilayer lipid membrane (tBLM), based on a (C14)2-(PEO)6-thiol anchor, WC14 [1]. Such membranes can be prepared to contain single phospholipids or complex lipid mixtures [2], including functional lipids involved in cell signaling, such as the highly charged phosphatidylinositol phosphates (PIPs). To study the interaction between the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) and model membranes we have incorporated phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in tBLMs and use fluorescence correlation spectroscopy (FCS), neutron reflectometry (NR) and surface plasmon resonance (SPR) for their characterization. NR shows that tBLMs formed with PI(4,5)P2 are complete. FCS of labeled PI(4,5)P2 shows that diffusion occurs at the time scale characteristic of membrane-incorporated lipid. Finally, SPR shows specific binding of PTEN to the model membrane thus confirming the incorporation of PI(4,5)P2 into the tBLM. [1] McGillivray et al, Biointerphases 2, 21-33 (2007) [2] Heinrich et al, Langmuir, submitted

  14. From lanosterol to cholesterol: structural evolution and differential effects on lipid bilayers.

    PubMed Central

    Miao, Ling; Nielsen, Morten; Thewalt, Jenifer; Ipsen, John H; Bloom, Myer; Zuckermann, Martin J; Mouritsen, Ole G

    2002-01-01

    Cholesterol is an important molecular component of the plasma membranes of mammalian cells. Its precursor in the sterol biosynthetic pathway, lanosterol, has been argued by Konrad Bloch (Bloch, K. 1965. Science. 150:19-28; 1983. CRC Crit. Rev. Biochem. 14:47-92; 1994. Blonds in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. Yale University Press, New Haven, CT.) to also be a precursor in the molecular evolution of cholesterol. We present a comparative study of the effects of cholesterol and lanosterol on molecular conformational order and phase equilibria of lipid-bilayer membranes. By using deuterium NMR spectroscopy on multilamellar lipid-sterol systems in combination with Monte Carlo simulations of microscopic models of lipid-sterol interactions, we demonstrate that the evolution in the molecular chemistry from lanosterol to cholesterol is manifested in the model lipid-sterol membranes by an increase in the ability of the sterols to promote and stabilize a particular membrane phase, the liquid-ordered phase, and to induce collective order in the acyl-chain conformations of lipid molecules. We also discuss the biological relevance of our results, in particular in the context of membrane domains and rafts. PMID:11867458

  15. Structure of the Disulfide Bond Generating Membrane Protein DsbB in the Lipid Bilayer

    PubMed Central

    Tang, Ming; Nesbitt, Anna E.; Sperling, Lindsay J.; Berthold, Deborah A.; Schwieters, Charles D.; Gennis, Robert B.; Rienstra, Chad M.

    2013-01-01

    The integral membrane protein DsbB in Escherichia coli is responsible for oxidizing the periplasmic protein DsbA, which forms disulfide bonds in substrate proteins. We have developed a high-resolution structural model by combining experimental X-ray and solid-state NMR with molecular dynamics (MD) simulations. We embedded the high-resolution DsbB structure, derived from the joint calculation with X-ray reflections and solid-state NMR restraints, into the lipid bilayer and performed MD simulations to provide a mechanistic view of DsbB function in the membrane. Further, we revealed the membrane topology of DsbB by selective proton spin diffusion experiments, which directly probe the correlations of DsbB with water and lipid acyl chains. NMR data also support the model of a flexible periplasmic loop and an interhelical hydrogen bond between Glu26 and Tyr153. PMID:23416557

  16. Elasticity, strength and stability of bilayer lipid membranes and their changes due to phospholipid modification.

    PubMed

    Passechnik, V I; Hianik, T; Karagodin, V P; Kagan, V E

    1984-12-01

    Elasticity measurements of bilayer lipid membranes (BLM) based on registration of the third harmonic of the membrane current during the application of a periodic tension to the membrane was used to study the effects of lipid peroxidation (LPO) and phospholipase A on BLM. LPO resulted in decreased values of the Young modulus for BLM, while some products of LPO and phospholipid hydrolysis (linolenic acid) were able to increase drastically the modulus. The presence of individual products of LPO and phospholipid hydrolysis in BLM produced non-additive effects on the elasticity, strength and stability of BLM. Lysolecithine strongly affected both the strength and stability of BLM. without changing its elasticity modulus. It was found that the lower the rate of structural changes in lecithine BLM, the longer its lifetime. Membranes having a heterogeneous polar composition form more stable BLM as compared to chemically homogeneous membranes.

  17. Spectroscopic study of 3-Hydroxyflavone - protein interaction in lipidic bi-layers immobilized on silver nanoparticles.

    PubMed

    Voicescu, Mariana; Ionescu, Sorana; Nistor, Cristina L

    2017-01-01

    The interaction of 3-Hydroxyflavone with serum proteins (BSA and HSA) in lecithin lipidic bi-layers (PC) immobilized on silver nanoparticles (SNPs), was studied by fluorescence and Raman spectroscopy. BSA secondary structure was quantified with a deconvolution algorithm, showing a decrease in α-helix structure when lipids were added to the solution. The effect of temperature on the rate of the excited-state intra-molecular proton transfer and on the dual fluorescence emission of 3-HF in the HSA/PC/SNPs systems was discussed. Evaluation of the antioxidant activity of 3-HF in HSA/PC/SNPs systems was also studied. The antioxidant activity of 3-HF decreased in the presence of SNPs. The results are discussed with relevance to the secondary structure of proteins and of the 3-HF based nano-systems to a topical formulation useful in the oxidative stress process. PMID:27380623

  18. Creating Biological Membranes on the Micron Scale: Forming Patterned Lipid Bilayers Using a Polymer Lift-Off Technique

    PubMed Central

    Orth, R. N.; Kameoka, J.; Zipfel, W. R.; Ilic, B.; Webb, W. W.; Clark, T. G.; Craighead, H. G.

    2003-01-01

    We present a new method for creating patches of fluid lipid bilayers with conjugated biotin and other compounds down to 1 μm resolution using a photolithographically patterned polymer lift-off technique. The patterns are realized as the polymer is mechanically peeled away in one contiguous piece in solution. The functionality of these surfaces is verified with binding of antibodies and avidin on these uniform micron-scale platforms. The biomaterial patches, measuring 1 μm–76 μm on edge, provide a synthetic biological substrate for biochemical analysis that is ∼100× smaller in width than commercial printing technologies. 100 nm unilamellar lipid vesicles spread to form a supported fluid lipid bilayer on oxidized silicon surface as confirmed by fluorescence photobleaching recovery. Fluorescence photobleaching recovery measurements of DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18(3))) stained bilayer patches yielded an average diffusion coefficient of 7.54 ± 1.25 μm2 s−1, equal to or slightly faster than typically found in DiI stained cells. This diffusion rate is ∼3× faster than previous values for bilayers on glass. This method provides a new means to form functionalized fluid lipid bilayers as micron-scale platforms to immobilize biomaterials, capture antibodies and biotinylated reagents from solution, and form antigenic stimuli for cell stimulation. PMID:14581207

  19. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane

    NASA Astrophysics Data System (ADS)

    Rajapaksha, Suneth P.; Pal, Nibedita; Zheng, Desheng; Lu, H. Peter

    2015-11-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ˜15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  20. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

    PubMed

    Rajapaksha, Suneth P; Pal, Nibedita; Zheng, Desheng; Lu, H Peter

    2015-01-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  1. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

    PubMed

    Rajapaksha, Suneth P; Pal, Nibedita; Zheng, Desheng; Lu, H Peter

    2015-01-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  2. Tethered bilayer lipid membranes studied by simultaneous attenuated total reflectance infrared spectroscopy and electrochemical impedance spectroscopy

    PubMed Central

    Erbe, Andreas; Bushby, Richard J.; Evans, Stephen D.; Jeuken, Lars J. C.

    2013-01-01

    The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self–assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO3Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance fourier transform infrared (ATR–FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicates that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO3Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, while spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long–living intermediates for surfaces of high 6-mercaptohexanol content. No long–living spherical vesicles have been detected for surfaces with large fraction of EO3Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation. PMID:17388505

  3. Long and Short Lipid Molecules Experience the Same Interleaflet Drag in Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Horner, Andreas; Akimov, Sergey A.; Pohl, Peter

    2013-06-01

    Membrane interleaflet viscosity ηe affects tether formation, phase separation into domains, cell shape changes, and budding. Contrary to the expected contribution to interleaflet coupling from interdigitation, the slide of lipid patches in opposing monolayers conferred the same value ηe≈3×109Jsm-4 for the friction experienced by the ends of both short and long chain fluorescent lipid analogues. Consistent with the weak dependence of the translational diffusion coefficient on lipid length, the in-layer viscosity was, albeit length dependent, much smaller than ηe.

  4. Effect of Lipid Bilayer on Human Islet Amyloid Polypeptide Self Assembly

    NASA Astrophysics Data System (ADS)

    Chiu, Chi-Cheng; Singh, Sadanand; de Pablo, Juan J.

    2012-02-01

    Aggregates of human islet amyloid polypeptides (hIAPP, also known as human amylin) are commonly found in the pancreatic β-cells of type II diabetes patients. Experimental studies have shown that small aggregates of hIAPP, that arise during the assembly process, lead to membrane leakage and are highly cytotoxic. Due to the fast assembly kinetics, it is difficult to study the early aggregation of hIAPP experimentally. In this work, we use molecular simulation with a coarse grained (CG) model to investigate the oligomerization of hIAPP with and without the presence of lipid bilayers. We develop a CG protein model that reproduces the three thremodynamically stable structures of hIAPP, namely α-helix, β-hairpin, and unstructured coil, and the corresponding free energy differences calculated by atomistic molecular simulations. The aggregated structure of hIAPP also agrees with that proposed by NMR experiments. We further investigate the assembly of hIAPP in the presence of a lipid bilayer and its effect on the membrane leakage. Comparing our results with the mechanism proposed based on experimental data provides a better understanding of the origins of hIAPP self assembly and its toxicity.

  5. On the optimal design of molecular sensing interfaces with lipid bilayer assemblies - A knowledge based approach

    NASA Astrophysics Data System (ADS)

    Siontorou, Christina G.

    2012-12-01

    Biosensors are analytic devices that incorporate a biochemical recognition system (biological, biologicalderived or biomimic: enzyme, antibody, DNA, receptor, etc.) in close contact with a physicochemical transducer (electrochemical, optical, piezoelectric, conductimetric, etc.) that converts the biochemical information, produced by the specific biological recognition reaction (analyte-biomolecule binding), into a chemical or physical output signal, related to the concentration of the analyte in the measuring sample. The biosensing concept is based on natural chemoreception mechanisms, which are feasible over/within/by means of a biological membrane, i.e., a structured lipid bilayer, incorporating or attached to proteinaceous moieties that regulate molecular recognition events which trigger ion flux changes (facilitated or passive) through the bilayer. The creation of functional structures that are similar to natural signal transduction systems, correlating and interrelating compatibly and successfully the physicochemical transducer with the lipid film that is self-assembled on its surface while embedding the reconstituted biological recognition system, and at the same time manage to satisfy the basic conditions for measuring device development (simplicity, easy handling, ease of fabrication) is far from trivial. The aim of the present work is to present a methodological framework for designing such molecular sensing interfaces, functioning within a knowledge-based system built on an ontological platform for supplying sub-systems options, compatibilities, and optimization parameters.

  6. /SIGMA PHI/-tocopherol: modifier of the phase state of the lipid bilayer

    SciTech Connect

    Skrypin, V.I.; Bratkcovskaya, L.B.; Erin, A.N.; Kagan, V.E.

    1985-05-01

    This paper determines the action of low (near-physiological) concentrations of alpha-tocopherol on the character of the gel-liquid crystal transition in a lipid bilayer containing free fatty acids. Fifty mM of K/sup +/-phosphate buffer was made up in D/sub 2/O and kept for several hours to enable substitution of H 1 by D 2, after which the buffer was dried and redissolved in D/sub 2/O. The graphs of temperature dependence of relative signal intensity of protons of methylene groups in liposomes of different composition are presented. It is shown that the stabilizing action of alpha-tocopherol on the phase state of the lipid bilayer of membranes is one of the mechanisms by which the recently demonstrated ability of alpha-tocopherol to protect biological membranes against the injurious action of free fatty acids, through the formation of complexes of alpha-tocopherol with fatty acides, may be effected.

  7. Voltage-sensitive styryl dyes as singlet oxygen targets on the surface of bilayer lipid membrane.

    PubMed

    Sokolov, V S; Gavrilchik, A N; Kulagina, A O; Meshkov, I N; Pohl, P; Gorbunova, Yu G

    2016-08-01

    Photosensitizers are widely used as photodynamic therapeutic agents killing cancer cells by photooxidation of their components. Development of new effective photosensitive molecules requires profound knowledge of possible targets for reactive oxygen species, especially for its singlet form. Here we studied photooxidation of voltage-sensitive styryl dyes (di-4-ANEPPS, di-8-ANEPPS, RH-421 and RH-237) by singlet oxygen on the surface of bilayer lipid membranes commonly used as cell membrane models. Oxidation was induced by irradiation of a photosensitizer (aluminum phthalocyanine tetrasulfonate) and monitored by the change of dipole potential on the surface of the membrane. We studied the drop of the dipole potential both in the case when the dye molecules were adsorbed on the same side of the lipid bilayer as the photosensitizer (cis-configuration) and in the case when they were adsorbed on the opposite side (trans-configuration). Based on a simple model, we determined the rate of oxidation of the dyes from the kinetics of change of the potential during and after irradiation. This rate is proportional to steady-state concentration of singlet oxygen in the membrane under irradiation. Comparison of the oxidation rates of various dyes reveals that compounds of ANEPPS series are more sensitive to singlet oxygen than RH type dyes, indicating that naphthalene group is primarily responsible for their oxidation. PMID:27236238

  8. Droplet immobilization within a polymeric organogel improves lipid bilayer durability and portability.

    PubMed

    Venkatesan, Guru A; Sarles, Stephen A

    2016-05-24

    The droplet interface bilayer (DIB) is a promising technique for assembling lipid membrane-based materials and devices using water droplets in oil, but it has largely been limited to laboratory environments due to its liquid construction. With a vision to transform this lab-based technique into a more-durable embodiment, we investigate the use of a polymer-based organogel to encapsulate DIBs within a more-solid material matrix to improve their handling and portability. Specifically, a temperature-sensitive organogel formed from hexadecane and poly[styrene-b-(ethylene-co-butylene)-b-styrene] (SEBS) triblock copolymer is used to replace the liquid solvent that surrounds the lipid-coated droplets to establish a novel liquid-in-gel DIB system. Through specific capacitance measurements and single-channel recordings of the pore forming peptide alamethicin, we verify that the structural and functional membrane properties are retained when DIBs are assembled within SEBS organogel. In addition, we demonstrate that organogel encapsulation offers improved handling of droplets and yields DIBs with a near 3× higher bilayer durability, as quantified by the lateral acceleration required to rupture the membrane, compared to liquid-in-liquid DIBs in oil. This encapsulated DIB system provides a barrier against contamination from the environment and offers a new material platform for supporting multilayered DIB-based devices as well as other digital microfluidic systems that feature water droplets in oil. PMID:27164314

  9. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers

    NASA Astrophysics Data System (ADS)

    Ashley, Carlee E.; Carnes, Eric C.; Phillips, Genevieve K.; Padilla, David; Durfee, Paul N.; Brown, Page A.; Hanna, Tracey N.; Liu, Juewen; Phillips, Brandy; Carter, Mark B.; Carroll, Nick J.; Jiang, Xingmao; Dunphy, Darren R.; Willman, Cheryl L.; Petsev, Dimiter N.; Evans, Deborah G.; Parikh, Atul N.; Chackerian, Bryce; Wharton, Walker; Peabody, David S.; Brinker, C. Jeffrey

    2011-05-01

    Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 106-fold improvement over comparable liposomes.

  10. Formation and fluidity measurement of supported lipid bilayer on polyvinyl chloride membrane

    SciTech Connect

    Kobayashi, Takuji Kono, Akiteru Sawada, Kazuaki; Futagawa, Masato; Tero, Ryugo

    2014-02-20

    We prepared an artificial lipid bilayer on a plasticized poly(vinyl chloride) (PVC) membrane on a Si3N4 layer deposited on a Si wafer. We optimized the experimental condition for the fabrication of the PVC membrane, and obtained a PVC membrane with a flat and uniform surface on the scale of several hundreds of micrometer suitable for a substrate for supported lipid bilayers (SLBs). The SLB of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was formed on the PVC membrane by the vesicle fusion method. The observation with a conventional epi-fluorescence microscope and a confocal laser scanning microscope gave geometrically uniform images of the SLB on the PVC membrane. The fluidity and the mobile fraction of the SLB was evaluated by the fluorescence recovery after photobleaching method, and compared with that on a thermally oxidized SiO{sub 2}/Si substrate. The SLB on the PVC membrane contained immobile fraction ∼30%, but the diffusion in the mobile fraction was two times faster than that in the SLB on SiO{sub 2}/Si, which had little immobile fraction.

  11. Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer.

    PubMed

    Liu, Lei; Xie, Jiani; Li, Ting; Wu, Hai-Chen

    2015-11-01

    We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage of the PROCEDURE is to cut and separate the SWCNTs. We cut long, purified SWCNTs with sonication in concentrated sulfuric acid/nitric acid (3/1). Isolation of ultrashort SWCNTs is carried out by size-exclusion HPLC separation. The second stage is to insert these short SWCNTs into the lipid bilayer. This step requires a microinjection probe made from a glass capillary. The setup for protein nanopore research can be adopted for the single-channel recording experiments without any special treatment. The obtained current traces are of very high quality, showing stable baselines and little background noise. Example procedures are shown for investigating ion transport and DNA translocation through these SWCNT nanopores. This nanopore has potential applications in molecular sensing, nanopore DNA sequencing and early disease diagnosis. For example, we have selectively detected modified 5-hydroxymethylcytosine in single-stranded DNA (ssDNA), which may have implications in screening specific genomic DNA sequences. The protocol takes ∼15 d, including SWCNT purification, cutting and separation, as well as the formation of SWCNT nanopores for DNA analyses.

  12. Reconstitution of Purified Acetylcholine Receptors with Functional Ion Channels in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Nelson, N.; Anholt, R.; Lindstrom, J.; Montal, M.

    1980-05-01

    Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16± 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35± 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.

  13. Direct measurements of heating by electromagnetically trapped gold nanoparticles on supported lipid bilayers.

    PubMed

    Bendix, Poul M; Reihani, S Nader S; Oddershede, Lene B

    2010-04-27

    Absorption of electromagnetic irradiation results in significant heating of metallic nanoparticles, an effect which can be advantageously used in biomedical contexts. Also, metallic nanoparticles are presently finding widespread use as handles, contacts, or markers in nanometer scale systems, and for these purposes it is essential that the temperature increase associated with electromagnetic irradiation is not harmful to the environment. Regardless of whether the heating of metallic nanoparticles is desired or not, it is crucial for nanobio assays to know the exact temperature increase associated with electromagnetic irradiation of metallic nanoparticles. We performed direct measurements of the temperature surrounding single gold nanoparticles optically trapped on a lipid bilayer, a biologically relevant matrix. The lipid bilayer had incorporated fluorescent molecules which have a preference for either fluid or gel phases. The heating associated with electromagnetic radiation was measured by visualizing the melted footprint around the irradiated particle. The effect was measured for individual gold nanoparticles of a variety of sizes and for a variety of laser powers. The temperatures were highly dependent on particle size and laser power, with surface temperature increments ranging from a few to hundreds of degrees Celsius. Our results show that by a careful choice of gold nanoparticle size and strength of irradiating electromagnetic field, one can control the exact particle temperature. The method is easily applicable to any type of nanoparticle for which the photothermal effect is sought to be quantified.

  14. Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity.

    PubMed

    Watanabe, Rikiya; Soga, Naoki; Fujita, Daishi; Tabata, Kazuhito V; Yamauchi, Lisa; Hyeon Kim, Soo; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Suga, Hiroaki; Noji, Hiroyuki

    2014-07-24

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins' function.

  15. Voltage-sensitive styryl dyes as singlet oxygen targets on the surface of bilayer lipid membrane.

    PubMed

    Sokolov, V S; Gavrilchik, A N; Kulagina, A O; Meshkov, I N; Pohl, P; Gorbunova, Yu G

    2016-08-01

    Photosensitizers are widely used as photodynamic therapeutic agents killing cancer cells by photooxidation of their components. Development of new effective photosensitive molecules requires profound knowledge of possible targets for reactive oxygen species, especially for its singlet form. Here we studied photooxidation of voltage-sensitive styryl dyes (di-4-ANEPPS, di-8-ANEPPS, RH-421 and RH-237) by singlet oxygen on the surface of bilayer lipid membranes commonly used as cell membrane models. Oxidation was induced by irradiation of a photosensitizer (aluminum phthalocyanine tetrasulfonate) and monitored by the change of dipole potential on the surface of the membrane. We studied the drop of the dipole potential both in the case when the dye molecules were adsorbed on the same side of the lipid bilayer as the photosensitizer (cis-configuration) and in the case when they were adsorbed on the opposite side (trans-configuration). Based on a simple model, we determined the rate of oxidation of the dyes from the kinetics of change of the potential during and after irradiation. This rate is proportional to steady-state concentration of singlet oxygen in the membrane under irradiation. Comparison of the oxidation rates of various dyes reveals that compounds of ANEPPS series are more sensitive to singlet oxygen than RH type dyes, indicating that naphthalene group is primarily responsible for their oxidation.

  16. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

    PubMed

    Ashley, Carlee E; Carnes, Eric C; Phillips, Genevieve K; Padilla, David; Durfee, Paul N; Brown, Page A; Hanna, Tracey N; Liu, Juewen; Phillips, Brandy; Carter, Mark B; Carroll, Nick J; Jiang, Xingmao; Dunphy, Darren R; Willman, Cheryl L; Petsev, Dimiter N; Evans, Deborah G; Parikh, Atul N; Chackerian, Bryce; Wharton, Walker; Peabody, David S; Brinker, C Jeffrey

    2011-05-01

    Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes. PMID:21499315

  17. The Targeted Delivery of Multicomponent Cargos to Cancer Cells via Nanoporous Particle-Supported Lipid Bilayers

    PubMed Central

    Ashley, Carlee E.; Carnes, Eric C.; Phillips, Genevieve K.; Padilla, David; Durfee, Paul N.; Brown, Page A.; Hanna, Tracey N.; Liu, Juewen; Phillips, Brandy; Carter, Mark B.; Carroll, Nick J.; Jiang, Xingmao; Dunphy, Darren R.; Willman, Cheryl L.; Petsev, Dimiter N.; Evans, Deborah G.; Parikh, Atul N.; Chackerian, Bryce; Wharton, Walker; Peabody, David S.; Brinker, C. Jeffrey

    2011-01-01

    Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability, and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma (HCC) exhibit a 10,000-fold greater affinity for HCC than for hepatocytes, endothelial cells, and immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, siRNA, and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer allow a single protocell loaded with a drug cocktail to kill a drug-resistant HCC cell, representing a 106-fold improvement over comparable liposomes. PMID:21499315

  18. Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer

    PubMed Central

    Simonsson Nyström, Lisa; Burazerovic, Sabina; Gunnarsson, Jenny; Snijder, Arjan; Geschwindner, Stefan

    2016-01-01

    Abstract Investigations of ligand‐binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity‐related limitations impaired by low signal‐to‐noise ratios. Inspired by affinity capturing of water‐soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane‐protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris‐NTA)–biotin conjugate. A polymer‐supported lipid bilayer containing His6‐tagged β‐secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6–BACE in the Ni2+–NTA‐modified region of the substrate resulted in a stationary three‐fold increase in surface coverage, and an accompanied increase in ligand‐binding response. PMID:27777836

  19. Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

    PubMed Central

    Watanabe, Rikiya; Soga, Naoki; Fujita, Daishi; Tabata, Kazuhito V.; Yamauchi, Lisa; Hyeon Kim, Soo; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Suga, Hiroaki; Noji, Hiroyuki

    2014-01-01

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function. PMID:25058452

  20. Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.

    PubMed

    Labarca, P; Lindstrom, J; Montal, M

    1984-04-01

    The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.

  1. Crowding-Induced Mixing Behavior of Lipid Bilayers: Examination of Mixing Energy, Phase, Packing Geometry, and Reversibility.

    PubMed

    Zeno, Wade F; Rystov, Alice; Sasaki, Darryl Y; Risbud, Subhash H; Longo, Marjorie L

    2016-05-10

    In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu(2+), chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10(-22) to 1.5 × 10(-20) J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. The mixing process was reversible, with Lo domains

  2. Crowding-induced mixing behavior of lipid bilayers: Examination of mixing energy, phase, packing geometry, and reversibility

    DOE PAGES

    Zeno, Wade F.; Rystov, Alice; Sasaki, Darryl Y.; Risbud, Subhash H.; Longo, Marjorie L.

    2016-04-20

    In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu2+, chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains ofmore » bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10–22 to 1.5 × 10–20 J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. Furthermore, the mixing process was reversible, with

  3. How Alcohol Chain-Length and Concentration Modulate Hydrogen Bond Formation in a Lipid Bilayer

    PubMed Central

    Dickey, Allison N.; Faller, Roland

    2007-01-01

    Molecular dynamics simulations are used to measure the change in properties of a hydrated dipalmitoylphosphatidylcholine bilayer when solvated with ethanol, propanol, and butanol solutions. There are eight oxygen atoms in dipalmitoylphosphatidylcholine that serve as hydrogen bond acceptors, and two of the oxygen atoms participate in hydrogen bonds that exist for significantly longer time spans than the hydrogen bonds at the other six oxygen atoms for the ethanol and propanol simulations. We conclude that this is caused by the lipid head group conformation, where the two favored hydrogen-bonding sites are partially protected between the head group choline and the sn-2 carbonyl oxygen. We find that the concentration of the alcohol in the ethanol and propanol simulations does not have a significant influence on the locations of the alcohol/lipid hydrogen bonds, whereas the concentration does impact the locations of the butanol/lipid hydrogen bonds. The concentration is important for all three alcohol types when the lipid chain order is examined, where, with the exception of the high-concentration butanol simulation, the alcohol molecules having the longest hydrogen-bonding relaxation times at the favored carbonyl oxygen acceptor sites also have the largest order in the upper chain region. The lipid behavior in the high-concentration butanol simulation differs significantly from that of the other alcohol concentrations in the order parameter, head group rotational relaxation time, and alcohol/lipid hydrogen-bonding location and relaxation time. This appears to be the result of the system being very near to a phase transition, and one occurrence of lipid flip-flop is seen at this concentration. PMID:17218462

  4. Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers*

    PubMed Central

    Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

    2012-01-01

    In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein. PMID:22139870

  5. Polymerized Planar Suspended Lipid Bilayers for Single Ion Channel Recordings: Comparison of Several Dienoyl Lipids

    PubMed Central

    Heitz, Benjamin A.; Xu, Juhua; Jones, Ian W.; Keogh, John P.; Comi, Troy J.; Hall, Henry K.; Aspinwall, Craig A.; Saavedra, S. Scott

    2011-01-01

    The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability to air-water transfers. BLM stability followed the order: bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α – hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single channel recordings of reconstituted ICs. PMID:21226498

  6. Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes

    SciTech Connect

    Yang, J.; Martí, J.; Calero, C.

    2014-03-14

    Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10{sup −5} cm{sup 2}/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10{sup −8} cm{sup 2}/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2–8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of

  7. Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes

    NASA Astrophysics Data System (ADS)

    Yang, J.; Calero, C.; Martí, J.

    2014-03-01

    Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10-5 cm2/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10-8 cm2/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2-8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of its interaction with lipids

  8. Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes.

    PubMed

    Yang, J; Calero, C; Martí, J

    2014-03-14

    Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10(-5) cm(2)/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10(-8) cm(2)/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2-8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of its interaction with

  9. The potassium channel KcsA and its interaction with the lipid bilayer.

    PubMed

    Williamson, I M; Alvis, S J; East, J M; Lee, A G

    2003-08-01

    The crystal structure of the K+ channel KcsA explains many features of ion channel function. The selectivity filter corresponds to a narrow region about 12 Along and 3 A wide, lined by carbonyl groups of the peptide backbone, through which a K+ ion can only move ina dehydrated form. The selectivity filter opens into a central, water-filled cavity leading to a gating site on the intracellular side of the channel. The channel is tetrameric, each monomer containing two transmembrane a helices, M1 and M2. Helix M1 faces the lipid bi-layer and helix M2 faces the central channel pore; the M2 helices participate in subunit-subunit interactions. Helices M1 and M2 in each subunit pack as a pair of antiparallel coils with a heptad repeat, but the M2 helices of neighbouring subunits show fewer interactions, crossing at an angle of about -40 degrees. Trp residues at the ends of the transmembrane a helices form clear girdles on the two faces of the membrane, which, together with girdles of charged residues, define a hydrophobic thickness of about 37 A for the channel. Binding constants for phosphatidylcholines to KcsA vary with fatty acyl chain length, the optimum chain length being C22. A phosphatidylcholine with this chain length gives a bilayer of thickness about 34 A in the liquid crystalline phase, matching the hydrophobic thickness of the protein. However, a typical biological membrane has a hydrophobic thickness of about 27 A. Thus either the transmembrane a helices of KcsA are more tilted in the native membrane than they are in the crystal structure, or the channel is under stress in the native membrane. The efficiency of hydrophobic matching between KcsA and the surrounding lipid bilayer is high over the chain length range C10-C24. The channel requires the presence of some anionic lipids for function, and fluorescence quenching studies show the presence of two classes of lipid binding site on KcsA; at one class of site (nonannular sites) anionic phospholipids bind more

  10. Characterization of the channel properties of tetanus toxin in planar lipid bilayers.

    PubMed Central

    Gambale, F; Montal, M

    1988-01-01

    A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less

  11. Derivation of coarse-grained simulation models of chlorophyll molecules in lipid bilayers for applications in light harvesting systems.

    PubMed

    Debnath, Ananya; Wiegand, Sabine; Paulsen, Harald; Kremer, Kurt; Peter, Christine

    2015-09-14

    The correct interplay of interactions between protein, pigment and lipid molecules is highly relevant for our understanding of the association behavior of the light harvesting complex (LHCII) of green plants. To cover the relevant time and length scales in this multicomponent system, a multi-scale simulation ansatz is employed that subsequently uses a classical all atomistic (AA) model to derive a suitable coarse grained (CG) model which can be backmapped into the AA resolution, aiming for a seamless conversion between two scales. Such an approach requires a faithful description of not only the protein and lipid components, but also the interaction functions for the indispensable pigment molecules, chlorophyll b and chlorophyll a (referred to as chl b/chl a). In this paper we develop a CG model for chl b and chl a in a dipalmitoylphosphatidyl choline (DPPC) bilayer system. The structural properties and the distribution behavior of chl within the lipid bilayer in the CG simulations are consistent with those of AA reference simulations. The non-bonded potentials are parameterized such that they fit to the thermodynamics based MARTINI force-field for the lipid bilayer and the protein. The CG simulation shows chl aggregation in the lipid bilayer which is supported by fluorescence quenching experiments. It is shown that the derived chl model is well suited for CG simulations of stable, structurally consistent, trimeric LHCII and can in the future be used to study their large scale aggregation behavior.

  12. Anisotropic solvent model of the lipid bilayer. 2. Energetics of insertion of small molecules, peptides, and proteins in membranes.

    PubMed

    Lomize, Andrei L; Pogozheva, Irina D; Mosberg, Henry I

    2011-04-25

    A new computational approach to calculating binding energies and spatial positions of small molecules, peptides, and proteins in the lipid bilayer has been developed. The method combines an anisotropic solvent representation of the lipid bilayer and universal solvation model, which predicts transfer energies of molecules from water to an arbitrary medium with defined polarity properties. The universal solvation model accounts for hydrophobic, van der Waals, hydrogen-bonding, and electrostatic solute-solvent interactions. The lipid bilayer is represented as a fluid anisotropic environment described by profiles of dielectric constant (ε), solvatochromic dipolarity parameter (π*), and hydrogen bonding acidity and basicity parameters (α and β). The polarity profiles were calculated using published distributions of quasi-molecular segments of lipids determined by neutron and X-ray scattering for DOPC bilayer and spin-labeling data that define concentration of water in the lipid acyl chain region. The model also accounts for the preferential solvation of charges and polar groups by water and includes the effect of the hydrophobic mismatch for transmembrane proteins. The method was tested on calculations of binding energies and preferential positions in membranes for small-molecules, peptides and peripheral membrane proteins that have been experimentally studied. The new theoretical approach was implemented in a new version (2.0) of our PPM program and applied for the large-scale calculations of spatial positions in membranes of more than 1000 peripheral and integral proteins. The results of calculations are deposited in the updated OPM database ( http://opm.phar.umich.edu ).

  13. Nanomechanical properties of lipid bilayer: Asymmetric modulation of lateral pressure and surface tension due to protein insertion in one leaflet of a bilayer

    NASA Astrophysics Data System (ADS)

    Maftouni, Negin; Amininasab, Mehriar; Ejtehadi, Mohammad Reza; Kowsari, Farshad; Dastvan, Reza

    2013-02-01

    The lipid membranes of living cells form an integral part of biological systems, and the mechanical properties of these membranes play an important role in biophysical investigations. One interesting problem to be evaluated is the effect of protein insertion in one leaflet of a bilayer on the physical properties of lipid membrane. In the present study, an all atom (fine-grained) molecular dynamics simulation is used to investigate the binding of cytotoxin A3 (CTX A3), a cytotoxin from snake venom, to a phosphatidylcholine lipid bilayer. Then, a 5-microsecond coarse-grained molecular dynamics simulation is carried out to compute the pressure tensor, lateral pressure, surface tension, and first moment of lateral pressure in each monolayer. Our simulations reveal that the insertion of CTX A3 into one monolayer results in an asymmetrical change in the lateral pressure and corresponding spatial distribution of surface tension of the individual bilayer leaflets. The relative variation in the surface tension of the two monolayers as a result of a change in the contribution of the various intermolecular forces may potentially be expressed morphologically.

  14. A self-consistent chain model for the phase transitions in lipid bilayer membranes.

    PubMed Central

    Kambara, T; Sasaki, N

    1984-01-01

    We presented a mechanical model of a lipid bilayer membrane. The internal conformations of a polar head group and double hydrocarbon chains in a lipid molecule were described on the basis of the isomeric bond-rotation scheme. The thermodynamic properties of the lipid membranes were represented by a density matrix that described the rotational isomeric states of the head groups and chains. The parameters that determined the density matrix were obtained in the presence of the intermolecular interactions, which depend on the conformation of the molecules. The interchain interaction was given by the Kihara potential, which depends on the shape of the chains. The Coulomb interaction between the polar head groups and the lateral pressure were considered. The calculation was made for the three lipid molecules corresponding to DMPC, DPPC, and DSPC. The model agreed well with the following experimental results: the temperature, the latent heat of the gel-to-liquid crystalline phase transition, the temperature dependencies of (a) the intermolecular distance, (b) the number of gauche bonds in a hydrocarbon chain, (c) the order parameter for the bond orientation, (d) the volume of the membrane, (e) the thermal expansion coefficients, and (f) the birefringence. PMID:6487736

  15. Comprehensive analysis of compositional interface fluctuations in planar lipid bilayer membranes.

    PubMed

    Han, Tao; Haataja, Mikko

    2011-11-01

    In this paper we present a comprehensive analysis of line tension-driven compositional interface fluctuations in planar lipid bilayer membranes. Our starting point is the advective Cahn-Hilliard equation for the local lipid composition in symmetric membranes, which explicitly incorporates both advective and diffusive lipid transport processes, and which is coupled to the continuum hydrodynamic equations governing the flow behavior of the membrane and surrounding solvent with finite subphase thickness. In order to extract the interface dynamics from the continuum phase-field formalism, we first derive the appropriate sharp-interface limit equations. We then carry out a linear perturbation analysis for the relaxational dynamics of small-amplitude sinusoidal interface fluctuations to yield the general dispersion relation ω(k) as a function of perturbation wave number k. The resulting expression incorporates the effects of diffusive and advective lipid transport processes within the membrane, viscous or viscoelastic membrane properties, coupling between membrane and solvent, and inertial effects within the membrane and solvent. It is shown that previously considered scenarios naturally emerge as limiting cases of the general result. Furthermore, we discuss two additional scenarios amenable to analysis, one in which the inertia of the solvent is relevant, and another one in which the membrane displays significant viscoelastic properties. Finally, we numerically evaluate the general dispersion relation for three representative model membrane systems. PMID:22181440

  16. Structures, dynamics, and water permeation free energy across bilayers of Lipid A and its analog studied with molecular dynamics simulation.

    PubMed

    Wei, Tao; Huang, Tiefan; Qiao, Baofu; Zhang, Mo; Ma, Heng; Zhang, Lin

    2014-11-20

    Fundamental studies of the supramolecular layer structures, dynamics and water permeation free energy of hexa-acyl-chain Lipid A and its analogue of tetra-acyl chains would be useful for polymer membranes design for endotoxin removal in water treatment, drug delivery and other biotechnologies. In this work, we studied their supramolecular bilayer by using molecular dynamics simulations and efficient free energy computations. Our simulation accuracy was verified by the agreement between the bilayer structural properties (structure factor, bilayer thickness, and the area per lipid) and lateral diffusion coefficient in our simulation and experimental measurements. More importantly, our simulation for the first time illustrated hexagonal compact packing of the hydrocarbon acyl chains within a leaflet of Lipid A membrane (at 298 K and water content of 40 wt %), which is consistent with experiments. In contrast, Lipid A analogue is found with less ordered ripple structures at the same condition. Our study also demonstrated slower dynamics and larger and broader free energy barrier (∼23 kJ/mol) for water permeation for Lipid A, compared with that of Lipid A analogue. Moreover, the analysis of dynamics showed that highly hydrated hydrophilic diglucosamine backbone is structurally stable, whereas the interdigitated hydrophobic acyl chain tails inside the membrane with faster dynamics screen the aqueous environment from the lipid interior and also reinforce the membrane's structural stability.

  17. How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

    PubMed Central

    Theodoulou, Frederica L.; Carrier, David J.; Schaedler, Theresia A.; Baldwin, Stephen A.; Baker, Alison

    2016-01-01

    Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data. PMID:27284041

  18. How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

    PubMed

    Theodoulou, Frederica L; Carrier, David J; Schaedler, Theresia A; Baldwin, Stephen A; Baker, Alison

    2016-06-15

    Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data. PMID:27284041

  19. Computer simulation of the distribution of hexane in a lipid bilayer: spatially resolved free energy, entropy, and enthalpy profiles.

    PubMed

    MacCallum, Justin L; Tieleman, D Peter

    2006-01-11

    The partitioning behavior of small molecules in lipid bilayers is important in a variety of areas including membrane protein folding and pharmacology. However, the inhomogeneous nature of lipid bilayers on a nanometer length scale complicates experimental studies of membrane partitioning. To gain more insight in the partitioning of a small molecule into the lipid bilayer, we have carried out atomistic computer simulations of hexane in a dioleoyl phosphatidylcholine model membrane. We have been able to obtain spatially resolved free energy, entropy, enthalpy, and heat capacity profiles based on umbrella sampling calculations at three different temperatures. In agreement with experiment, hexane partitions preferentially to the center of the bilayer. This process is driven almost entirely by a favorable entropy change, consistent with the hydrophobic effect. In contrast, partitioning to the densest region of the acyl chains is dominated by a favorable enthalpy change with a small entropy change, which is consistent with the "nonclassical" hydrophobic effect or "bilayer" effect. We explain the features of the entropy and enthalpy profiles in terms of density and free volume in the system. PMID:16390139

  20. Experimental study of the bending elasticity of charged lipid bilayers in aqueous solutions with pH5

    NASA Astrophysics Data System (ADS)

    Mitkova, D.; Stoyanova-Ivanova, A.; Ermakov, Yu A.; Vitkova, V.

    2012-12-01

    Exposure to high concentrations of contaminations due to air polluting gases, vapours and aerosols and possibly altering the normal pH in the body could lead to undesirable changes in the properties of biological cells. Here, we study experimentally the mechanical properties of synthetic phospholipid bilayers containing increasing molar fractions (up to 0.15) of charged lipid (synthetic phosphatidylserine) in aqueous solutions with controlled ionic strength and at pH 5, which is slightly lower than the physiological values of pH. Our observations in phase contrast and fluorescence testified to the coexistence of two phases in membranes for temperatures below 29°C. Micro-sized inhomogeneities in vesicle membranes were systematically observed at temperatures lower than 29°C and for molar fractions of phosphatidylserine in the bilayer higher than 0.1. For the quantitative determination of the membrane bending rigidity, we applied thermal fluctuation analysis of the shape of quasispherical lipid vesicles. As far as the liquid-crystalline state of the bilayer is a necessary condition for the application of the experimental method, only vesicles satisfying this requirement were processed for determination of their membrane bending rigidity. The value obtained for the bending modulus of bilayers with 0.15 molar content of charged lipid is about two times higher than the bending modulus of uncharged membranes in the same bathing solution. These findings are in qualitative agreement with our previous results for the bending rigidity of charged bilayers, measured by vesicle micromanipulation.

  1. The effects of ethylene oxide containing lipopolymers and tri-block copolymers on lipid bilayers of dipalmitoylphosphatidylcholine.

    PubMed Central

    Baekmark, T R; Pedersen, S; Jørgensen, K; Mouritsen, O G

    1997-01-01

    A comparative study is conducted on the influence of two types of polymeric compounds on the phase behavior of 1,2-dihexadecanoyl-s,n-glycero-3-phosphotidylcholine (DC16PC) lipid bilayers. The first polymeric compound is a lipopolymer, with two different lengths of a hydrophilic polyethylene oxide moity, anchored to the bilayer by a 1,2-dioctadecanoyl-s,n-glycero-3-phosphoethanolamine (DC18PE) lipid. The second type, which is a novel type of membrane-spanning object, is an amphiphilic tri-block copolymer composed of two hydrophilic stretches of polyethylene oxide separated by a hydrophobic stretch of polystyrene. Hence the tri-block copolymer may act as a membrane-spanning macromolecule mimicking an amphiphilic protein or polypeptide. Differential scanning calorimetry is used to determine a partial phase diagram for the lipopolymer systems and to assess the amount of lipopolymer that can be loaded into DC16PC lipid bilayers before micellization takes place. Unilamellar and micellar phase structures are investigated by fluorescence quenching using bilayer permeating dithionite. The chain length-dependent critical lipopolymer concentration, denoting the lamellar-to-micellar phase transition, compares favorably with a theoretical prediction based on free-energy considerations involving bilayer cohesion and lateral pressure exerted by the polymer chains. Images FIGURE 10 PMID:9284315

  2. Characterization of ion channels from Acetabularia plasma membrane in planar lipid bilayers.

    PubMed

    White, P J; Smahel, M; Thiel, G

    1993-04-01

    Plasma membrane from Acetabularia acetabulum was prepared by aqueous-polymer two-phase partitioning and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers by stirring in the presence of a (cis:trans) 325:100 mM KCl gradient. Under these conditions five distinct K(+)-selective channels were observed which had unitary chord-conductances (determined between 30 mV either side of the reversal potential) and frequencies of incorporation (in parentheses) of 1,600 pS (26%), 485 pS (21%), 259 pS (53%), 140 pS (37%) and 27 pS (37%). Two Cl(-)-selective channels were also observed, which had unitary chord-conductances of 8 and 48 pS and were present in 21 and 16% of bilayers, respectively. The voltage dependencies of channel open probability (Po), open-state time constant (tau o) and closed-state time constant (tau c) were determined for the 259, 140 and 27 pS K+ channels. The Po of all three channels increased with increasingly positive membrane potentials. Thus, since these channels were oriented with their extracellular face adjacent to the cis chamber, which was grounded, all would exhibit outward rectification in vivo. Changes in Po were effected by modulation of tau c in all channels, which shortened as membrane potentials became more positive, and also of tau o in the 140 and 27 pS channels, which increased as membrane potentials became more positive. Extracellular (cis) KCl concentration (and/or the KCl gradient across the bilayer) affected the Po of all three K+ channels, shifting the Po/membrane potential relationship in the direction of the change in the potassium reversal potential. In all channels this was achieved largely by changes in tau c.

  3. Probing Biological Processes on Supported Lipid Bilayers with Single-Walled Carbon Nanotube Field-Effect Transistors

    NASA Astrophysics Data System (ADS)

    Zhou, Xinjian; Moran-Mirabal, Jose Manuel; Craighead, Harold; McEuen, Paul

    2006-03-01

    We have formed supported lipid bilayers (SLBs) by small unilamellar vesicle fusion on substrates containing single-walled carbon nanotube field-effect transistors (SWNT-FETs). We are able to detect the self-assembly of SLBs electrically with SWNT-FETs since their threshold voltages are shifted by this event. The SLB fully covers the NT surface and lipid molecules can diffuse freely in the bilayer surface across the NT. To study the interactions of important biological entities with receptors imbedded within the membrane, we have also integrated a membrane protein, GT1b ganglioside, in the bilayer. While bare gangliosides can diffuse freely across the NT, interestingly the NT acts as a diffusion barrier for the gangliosides when they are bound with tetanus toxin. This experiment opens the possibility of using SWNT-FETs as biosensors for label-free detection.

  4. Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

    PubMed

    Hatty, Claire R; Le Brun, Anton P; Lake, Vanessa; Clifton, Luke A; Liu, Guo Jun; James, Michael; Banati, Richard B

    2014-03-01

    The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

  5. A New Route to Liposil Formation by an Interfacial Sol-Gel Process Confined by Lipid Bilayer.

    PubMed

    Shen, Shukun; Yang, Lu; Lu, Yaxing; Chen, Jian-Gang; Song, Shaofei; Hu, Daodao; Parikh, Atul

    2015-11-18

    We report a new and simple approach to prepare a class of silica-reinforced liposomes with hybrid core-shell nanostructures. The amphiphilic natural structure of lipids was exploited to sequester hydrophobic molecules, namely precursor TEOS and pyrene, in the hydrophobic midplane of liposomal bilayer assemblies in the aqueous phase. Subsequent interfacial hydrolysis of TEOS at the bilayer/water interface and ensuing condensation within the hydrophobic interstices of the lipid bilayer drives silica formation in situ, producing a novel class of silica-lipid hybrid liposils. Structural characterization by scanning- and transmission electron microscopy confirm that the liposils so generated preserve closed topologies and size-monodipersity of the parent lecithin liposomes, and DSC-TGA and XRD measurements provide evidence for the silica coating. Monitoring fluorescence measurements using embedded pyrene yield detailed information on microenvironment changes, which occur during sol-gel process and shed light on the structural evolution during silica formation. We envisage that liposils formed by this simple, new approach, exploiting the hydrophobic core of the lipid bilayer to spatially localize silica-forming precursors enables preparation of stable liposils exhibiting capacity for cargo encapsulation, bicompatibility, and fluorescence monitoring, more generally opening a window for construction of stable, functional hybrid materials. PMID:26197062

  6. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers.

    PubMed Central

    Oliver, A E; Deamer, D W

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two

  7. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

    NASA Technical Reports Server (NTRS)

    Oliver, A. E.; Deamer, D. W.

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two

  8. Modelling of noble anaesthetic gases and high hydrostatic pressure effects in lipid bilayers

    DOE PAGES

    Moskovitz, Yevgeny; Yang, Hui

    2015-01-08

    Our objective was to study molecular processes that might be responsible for inert gas narcosis and high-pressure nervous syndrome. The classical molecular dynamics trajectories (200 ns-long) of dioleoylphosphatidylcholine (DOPC) bilayers simulated by the Berger force field were evaluated for water and the atomic distribution of noble gases around DOPC molecules at a pressure range of 1 - 1000 bar and temperature of 310 Kelvin. Xenon and argon have been tested as model gases for general anesthetics, and neon has been investigated for distortions that are potentially responsible for neurological tremor at hyperbaric conditions. The analysis of stacked radial pair distributionmore » functions of DOPC headgroup atoms revealed the explicit solvation potential of gas molecules, which correlates with their dimensions. The orientational dynamics of water molecules at the biomolecular interface should be considered as an influential factor; while excessive solvation effects appearing in the lumen of membrane-embedded ion channels could be a possible cause of inert gas narcosis. All the noble gases tested exhibit similar patterns of the order parameter for both DOPC acyl chains, which is opposite to the patterns found for the order parameter curve at high hydrostatic pressures in intact bilayers. This finding supports the ‘critical volume’ hypothesis of anesthesia pressure reversal. The irregular lipid headgroup-water boundary observed in DOPC bilayers saturated with neon in the pressure range of 1 - 100 bar could be associated with the possible manifestation of neurological tremor at the atomic scale. The non-immobilizer neon also demonstrated the highest momentum impact on the normal component of the DOPC diffusion coefficient representing monolayers undulations rate, which indicates enhanced diffusivity, rather than atom size, as the key factor.« less

  9. Modelling of noble anaesthetic gases and high hydrostatic pressure effects in lipid bilayers

    SciTech Connect

    Moskovitz, Yevgeny; Yang, Hui

    2015-01-08

    Our objective was to study molecular processes that might be responsible for inert gas narcosis and high-pressure nervous syndrome. The classical molecular dynamics trajectories (200 ns-long) of dioleoylphosphatidylcholine (DOPC) bilayers simulated by the Berger force field were evaluated for water and the atomic distribution of noble gases around DOPC molecules at a pressure range of 1 - 1000 bar and temperature of 310 Kelvin. Xenon and argon have been tested as model gases for general anesthetics, and neon has been investigated for distortions that are potentially responsible for neurological tremor at hyperbaric conditions. The analysis of stacked radial pair distribution functions of DOPC headgroup atoms revealed the explicit solvation potential of gas molecules, which correlates with their dimensions. The orientational dynamics of water molecules at the biomolecular interface should be considered as an influential factor; while excessive solvation effects appearing in the lumen of membrane-embedded ion channels could be a possible cause of inert gas narcosis. All the noble gases tested exhibit similar patterns of the order parameter for both DOPC acyl chains, which is opposite to the patterns found for the order parameter curve at high hydrostatic pressures in intact bilayers. This finding supports the ‘critical volume’ hypothesis of anesthesia pressure reversal. The irregular lipid headgroup-water boundary observed in DOPC bilayers saturated with neon in the pressure range of 1 - 100 bar could be associated with the possible manifestation of neurological tremor at the atomic scale. The non-immobilizer neon also demonstrated the highest momentum impact on the normal component of the DOPC diffusion coefficient representing monolayers undulations rate, which indicates enhanced diffusivity, rather than atom size, as the key factor.

  10. Modelling of noble anaesthetic gases and high hydrostatic pressure effects in lipid bilayers.

    PubMed

    Moskovitz, Yevgeny; Yang, Hui

    2015-03-21

    Our objective was to study molecular processes that might be responsible for inert gas narcosis and high-pressure nervous syndrome. The classical molecular dynamics trajectories (200 ns) of dioleoylphosphatidylcholine (DOPC) bilayers simulated by the Berger force field were evaluated for water and the atomic distribution of noble gases around DOPC molecules in the pressure range of 1-1000 bar and at a temperature of 310 K. Xenon and argon have been tested as model gases for general anaesthetics, and neon has been investigated for distortions that are potentially responsible for neurological tremors in hyperbaric conditions. The analysis of stacked radial pair distribution functions of DOPC headgroup atoms revealed the explicit solvation potential of the gas molecules, which correlates with their dimensions. The orientational dynamics of water molecules at the biomolecular interface should be considered as an influential factor, while excessive solvation effects appearing in the lumen of membrane-embedded ion channels could be a possible cause of inert gas narcosis. All the noble gases tested exhibit similar order parameter patterns for both DOPC acyl chains, which are opposite of the patterns found for the order parameter curve at high hydrostatic pressures in intact bilayers. This finding supports the 'critical volume' hypothesis of anaesthesia pressure reversal. The irregular lipid headgroup-water boundary observed in DOPC bilayers saturated with neon in the pressure range of 1-100 bar could be associated with the possible manifestation of neurological tremors at the atomic scale. The non-immobiliser neon also demonstrated the highest momentum impact on the normal component of the DOPC diffusion coefficient representing the monolayer undulation rate, which indicates that enhanced diffusivity rather than atomic size is the key factor. PMID:25612767

  11. Experimental and Monte Carlo simulation studies of the thermodynamics of polyethyleneglycol chains grafted to lipid bilayers.

    PubMed Central

    Rex, S; Zuckermann, M J; Lafleur, M; Silvius, J R

    1998-01-01

    Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a "mushroom" regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for

  12. Polystyrene nanoparticle exposure induces ion-selective pores in lipid bilayers

    PubMed Central

    Negoda, Alexander; Kim, Kwang-Jin; Crandall, Edward D.; Worden, Robert M.

    2014-01-01

    A diverse range of molecular interactions can occur between engineered nanomaterials (ENM) and biomembranes, some of which could lead to toxic outcomes following human exposure to ENM. In this study, we adapted electrophysiology methods to investigate the ability of 20 nm polystyrene nanoparticles (PNP) to induce pores in model bilayer lipid membranes (BLM) that mimic biomembranes. PNP charge was varied using PNP decorated with either positive (amidine) groups or negative (carboxyl) groups, and BLM charge was varied using dioleoyl phospholipids having cationic (ethylphosphocholine), zwitterionic (phosphocholine), or anionic (phosphatidic acid) headgroups. Both positive and negative PNP induced BLM pores for all lipid compositions studied, as evidenced by current spikes and integral conductance. Stable PNP-induced pores exhibited ion selectivity, with the highest selectivity for K+ (PK/PCl ~ 8.3) observed when both the PNP and lipids were negatively charged, and the highest selectivity for Cl− (PK/PCl ~ 0.2) observed when both the PNP and lipids were positively charged. This trend is consistent with the finding that selectivity for an ion in channel proteins is imparted by oppositely charged functional groups within the channel’s filter region. The PK/PCl value was unaffected by the voltage-ramp method, the pore conductance, or the side of the BLM to which the PNP were applied. These results demonstrate for the first time that PNP can induce ion-selective pores in BLM, and that the degree of ion selectivity is influenced synergistically by the charges of both the lipid headgroups and functional groups on the PNP. PMID:23747366

  13. Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer

    PubMed Central

    Grudzinski, Wojciech; Sagan, Joanna; Welc, Renata; Luchowski, Rafal; Gruszecki, Wieslaw I.

    2016-01-01

    Amphotericin B is a popular antifungal antibiotic, a gold standard in treatment of systemic mycotic infections, due to its high effectiveness. On the other hand, applicability of the drug is limited by its considerable toxicity to patients. Biomembranes are a primary target of physiological activity of amphotericin B and both the pharmacologically desired and toxic side effects of the drug relay on its molecular organization in the lipid phase. In the present work, molecular organization, localization and orientation of amphotericin B, in a single lipid bilayer system, was analysed simultaneously, thanks to application of a confocal fluorescence lifetime imaging microscopy of giant unilamellar vesicles. The results show that the presence of sterols, in the lipid phase, promotes formation of supramolecular structures of amphotericin B and their penetration into the membrane hydrophobic core. The fact that such an effect is substantially less pronounced in the case of cholesterol than ergosterol, the sterol of fungal membranes, provides molecular insight into the selectivity of the drug. PMID:27620838

  14. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

    NASA Astrophysics Data System (ADS)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-01

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  15. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

    PubMed

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-28

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  16. Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer.

    PubMed

    Grudzinski, Wojciech; Sagan, Joanna; Welc, Renata; Luchowski, Rafal; Gruszecki, Wieslaw I

    2016-01-01

    Amphotericin B is a popular antifungal antibiotic, a gold standard in treatment of systemic mycotic infections, due to its high effectiveness. On the other hand, applicability of the drug is limited by its considerable toxicity to patients. Biomembranes are a primary target of physiological activity of amphotericin B and both the pharmacologically desired and toxic side effects of the drug relay on its molecular organization in the lipid phase. In the present work, molecular organization, localization and orientation of amphotericin B, in a single lipid bilayer system, was analysed simultaneously, thanks to application of a confocal fluorescence lifetime imaging microscopy of giant unilamellar vesicles. The results show that the presence of sterols, in the lipid phase, promotes formation of supramolecular structures of amphotericin B and their penetration into the membrane hydrophobic core. The fact that such an effect is substantially less pronounced in the case of cholesterol than ergosterol, the sterol of fungal membranes, provides molecular insight into the selectivity of the drug. PMID:27620838

  17. Simulation of fusion-mediated nanoemulsion interactions with model lipid bilayers

    PubMed Central

    Lee, Sun-Joo; Schlesinger, Paul H.; Wickline, Samuel A.; Lanza, Gregory M.; Baker, Nathan A.

    2012-01-01

    Perfluorocarbon-based nanoemulsion particles have become promising platforms for the delivery of therapeutic and diagnostic agents to specific target cells in a non-invasive manner. A “contact-facilitated” delivery mechanism has been proposed wherein the emulsifying phospholipid monolayer on the nanoemulsion surface contacts and forms a lipid complex with the outer monolayer of target cell plasma membrane, allowing cargo to diffuse to the surface of target cell. While this mechanism is supported by experimental evidence, its molecular details are unknown. The present study develops a coarse-grained model of nanoemulsion particles that are compatible with the MARTINI force field. Simulations using this coarse-grained model have demonstrated multiple fusion events between the particles and a model vesicular lipid bilayer. The fusion proceeds in the following sequence: dehydration at the interface, close apposition of the particles, protrusion of hydrophobic molecules to the particle surface, transient lipid complex formation, absorption of nanoemulsion into the liposome. The initial monolayer disruption acts as a rate-limiting step and is strongly influenced by particle size as well as by the presence of phospholipids supporting negative spontaneous curvature. The core-forming perfluorocarbons play critical roles in initiating the fusion process by facilitating protrusion of hydrophobic moieties into the interface between the two particles. This study directly supports the hypothesized nanoemulsion delivery mechanism and provides the underlying molecular details that enable engineering of nanoemulsions for a variety of medical applications. PMID:22712024

  18. Adsorption of α-Synuclein on Lipid Bilayers: Modulating the Structure and Stability of Protein Assemblies

    PubMed Central

    Haque, Farzin; Pandey, Anjan P.; Cambrea, Lee R.; Rochet, Jean-Christophe; Hovis, Jennifer S.

    2010-01-01

    The interaction of α-synuclein with phospholipid membranes has been examined using supported lipid bilayers and epi-fluorescence microscopy. The membranes contained phosphatidylcholine (PC) and phosphatidic acid (PA), which mix at physiological pH. Upon protein adsorption the lipids undergo fluid-fluid phase separation into PC-rich and PA-rich regions. The protein preferentially adsorbs to the PA-rich regions. The adsorption and subsequent aggregation of α-synuclein was probed by tuning several parameters: the charge on the lipids, the charge on the protein, and the screening environment. Conditions which promoted the greatest extent of adsorption resulted in structurally heterogeneous aggregates, while comparatively homogeneous aggregates were observed under conditions whereby adsorption did not occur as readily. Our observation that different alterations to the system lead to different degrees of aggregation and different aggregate structures poses a challenge for drug discovery. Namely, therapies aimed at neutralizing α-synuclein must target a broad range of potentially toxic, membrane-bound assemblies. PMID:20187615

  19. Stimulation-dependent gating of TRPM3 channel in planar lipid bilayers.

    PubMed

    Uchida, Kunitoshi; Demirkhanyan, Lusine; Asuthkar, Swapna; Cohen, Alejandro; Tominaga, Makoto; Zakharian, Eleonora

    2016-03-01

    The transient receptor potential melastatin (TRPM)-3 channel is critical for various physiologic processes. In somatosensory neurons, TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, whereas in pancreatic β-cells the channel has been linked to glucose-induced insulin release. As a typical representative of the TRP family, TRPM3 is highly polymodal. In cells, it is activated by heat and chemical agonists, including pregnenolone sulfate (PS) and nifedipine (Nif). To define the nuances of TRPM3 channel activity and its modulators, we succeeded in incorporating the TRPM3 protein into planar lipid bilayers. We found that phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole is necessary for channel opening by PS. Unlike PS, the presence of Nif alone sufficed to induce TRPM3 activity and demonstrated distinct gating behavior. We also performed an extensive thermodynamic analysis of TRPM3 activation and found that TRPM3 exhibited slight temperature sensitivity in the bilayers. In the absence of other agonists TRPM3 channels remained closed upon heat-induced stimulation, but opened in the presence of PIP2, although with only a low open-probability profile. Together, our results elucidate the details peculiar to TRPM3 channel function in an isolated system. We confirmed its direct gating by PS and PIP2, but found a lack of the strong intrinsic temperature sensitivity common to other thermosensitive TRP channels.

  20. Surface Dilution Kinetics of Phospholipase A2 Catalyzed Lipid-Bilayer Hydrolysis

    PubMed Central

    Singh, Jasmeet; Ranganathan, Radha

    2014-01-01

    Phospholipase A2 (PLA2) enzymes catalyze hydrolysis of phospholipids in membranes. Elucidation of the kinetics of interfacial enzymatic activity is best accomplished by investigating the interface substrate concentration dependence of the activity, for which appropriate diluents are required. PLA2 is stereo selective toward the L_enantiomers of phospholipids. A novel approach employing D_phospholipids as diluents to perform surface dilution kinetic studies of PLA2 is presented. Activity of bee-venom PLA2 at mixed L+D_DPPC (dipalmitoylphosphatidylcholine) bilayer interfaces was measured as functions of substrate L_DPPC mole fraction and vesicle concentration, using a sensitive fluorescence assay. A model for interface enzymatic activity based on the three-step kinetic scheme of: (i) binding of PLA2 to the bilayer interface; (ii) binding of a lipid to PLA2 at the interface; and (iii) hydrolysis, was applied to the hydrolysis data. Activity profiles showed that D_enantiomers also bind to the enzyme but resist hydrolysis. Activity dependences on vesicle and substrate concentrations could be disentangled, bringing resolution to an outstanding problem in membrane hydrolysis, of separating the effects of the three steps. Individual values of the kinetic parameters of the model including the vesicle-PLA2 equilibrium dissociation constant of step (i), interface Michaelis-Menten-Henri constant for L and D_DPPC of step (ii), and the rate constant for interface hydrolysis, step (iii) were obtained as solutions to equations resulting from fitting the model to the data. PMID:24491041

  1. Polyelectrolyte-Mediated Transport of Doxorubicin Through the Bilayer Lipid Membrane

    NASA Astrophysics Data System (ADS)

    Yaroslavov, Alexander A.; Kitaeva, Marina V.; Melik-Nubarov, Nikolay S.; Menger, Frederic M.

    A model is developed for the effect of ionic polymers on the transport of doxorubicin, an antitumor drug, through a bilayer membrane. Accordingly, a protonated (cationic) form of doxorubicin binds to an anionic polymer, poly(acrylic acid), the resulting complex being several hundred nanometers in size. Nevertheless, large complex species associate with neutral egg lecithin liposomes by means of hydrophobic attraction between the doxorubicin and the liposome bilayer. Then, the doxorubicin enters the liposome interior which has been imparted with an acidic buffer to protonate the doxorubicin. The rate of transmembrane Dox permeation decreases when elevating the polyacid-to-doxorubicin ratio. A cationic polymer, polylysine, being coupled with liposomes containing the negative lipid cardiolipin, accelerates membrane transport of doxorubicin with the maximum rate at a complete neutralization of the membrane charge by an interacting polycation. The effect of a polycation on doxorubicin transport becomes more pronounced as small negative liposomes (60-80 nm in diameter) are changed to larger ones (approx. 600 nm in diameter). An opportunity thus opens up for the manipulation of the kinetics of drug uptake by cells and, ultimately, the control of the pharmaceutical action of drugs.

  2. Does fluoride disrupt hydrogen bond network in cationic lipid bilayer? Time-dependent fluorescence shift of Laurdan and molecular dynamics simulations.

    PubMed

    Pokorna, Sarka; Jurkiewicz, Piotr; Vazdar, Mario; Cwiklik, Lukasz; Jungwirth, Pavel; Hof, Martin

    2014-12-14

    Time-dependent fluorescence shift (TDFS) of Laurdan embedded in phospholipid bilayers reports on hydration and mobility of the phospholipid acylgroups. Exchange of H2O with D2O prolongs the lifetime of lipid-water and lipid-water-lipid interactions, which is reflected in a significantly slower TDFS kinetics. Combining TDFS measurements in H2O and D2O hydrated bilayers with atomistic molecular dynamics (MD) simulations provides a unique tool for characterization of the hydrogen bonding at the acylgroup level of lipid bilayers. In this work, we use this approach to study the influence of fluoride anions on the properties of cationic bilayers composed of trimethylammonium-propane (DOTAP). The results obtained for DOTAP are confronted with those for neutral phosphatidylcholine (DOPC) bilayers. Both in DOTAP and DOPC H2O/D2O exchange prolongs hydrogen-bonding lifetime and does not disturb bilayer structure. These results are confirmed by MD simulations. TDFS experiments show, however, that for DOTAP this effect is cancelled in the presence of fluoride ions. We interpret these results as evidence that strongly hydrated fluoride is able to steal water molecules that bridge lipid carbonyls. Consequently, when attracted to DOTAP bilayer, fluoride disrupts the local hydrogen-bonding network, and the differences in TDFS kinetics between H2O and D2O hydrated bilayers are no longer observed. A distinct behavior of fluoride is also evidenced by MD simulations, which show different lipid-ion binding for Cl(-) and F(-).

  3. The binding and insertion of imidazolium-based ionic surfactants into lipid bilayers: the effects of the surfactant size and salt concentration.

    PubMed

    Lee, Hwankyu; Jeon, Tae-Joon

    2015-02-28

    Imidazolium-based ionic surfactants with hydrocarbon tails of different sizes were simulated with lipid bilayers at different salt concentrations. Starting with the random position of ionic surfactants outside the bilayer, surfactants with long tails mostly insert into the bilayer, while those with short tails show the insertion of fewer surfactant molecules, indicating the effect of the tail length. In particular, surfactants with a tail of two or four hydrocarbons insert and reversibly detach from the bilayer, while the inserted longer surfactants cannot be reversibly detached because of the strong hydrophobic interaction with lipid tails, in quantitative agreement with experiments. Longer surfactants insert more deeply and irreversibly into the bilayer and thus increase lateral diffusivities of the bilayer, indicating that longer surfactants more significantly disorder lipid bilayers, which also agrees with experiments regarding the effect of the tail length of ionic surfactants on membrane permeability and toxicity. Addition of NaCl ions weakens the electrostatic interactions between headgroups of surfactants and lipids, leading to the binding of fewer surfactants into the bilayer. In particular, our simulation findings indicate that insertion of ionic surfactants can be initiated by either the hydrophobic interaction between tails of surfactants and lipids or the electrostatic binding between imidazolium heads and lipid heads, and the strength of hydrophobic and electrostatic interactions depends on the tail length of surfactants.

  4. MD Simulations of P-Type ATPases in a Lipid Bilayer System.

    PubMed

    Autzen, Henriette Elisabeth; Musgaard, Maria

    2016-01-01

    Molecular dynamics (MD) simulation is a computational method which provides insight on protein dynamics with high resolution in both space and time, in contrast to many experimental techniques. MD simulations can be used as a stand-alone method to study P-type ATPases as well as a complementary method aiding experimental studies. In particular, MD simulations have proved valuable in generating and confirming hypotheses relating to the structure and function of P-type ATPases. In the following, we describe a detailed practical procedure on how to set up and run a MD simulation of a P-type ATPase embedded in a lipid bilayer using software free of use for academics. We emphasize general considerations and problems typically encountered when setting up simulations. While full coverage of all possible procedures is beyond the scope of this chapter, we have chosen to illustrate the MD procedure with the Nanoscale Molecular Dynamics (NAMD) and the Visual Molecular Dynamics (VMD) software suites.

  5. Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms.

    PubMed

    Weichbrodt, Conrad; Bajaj, Harsha; Baaken, Gerhard; Wang, Jiajun; Guinot, Serap; Kreir, Mohamed; Behrends, Jan C; Winterhalter, Mathias; Fertig, Niels

    2015-07-21

    In general, the method of choice to characterize the conductance properties of channel-forming bacterial porins is electrophysiology. Here, the classical method is to reconstitute single porins into planar lipid bilayers to derive functional information from the observed channel conductance. In addition to an estimated pore size, ion selectivity or transport properties in general are of importance. For the latter, measuring the ion current fluctuation can provide some information about the mode of transport of charged molecules penetrating the proteins. For instance, increasing the external voltage modifies the residence time in the channel: charged molecules with the ability to permeate through channels will travel faster whereas non-permeating molecules get pushed to the constriction zone with enhanced residence time. Here, we are interested in the ability of antibiotics to permeate channels and compare different techniques to reveal fast events.