Evidence of femtosecond-laser pulse induced cell membrane nanosurgery

NASA Astrophysics Data System (ADS)

Katchinskiy, Nir; Godbout, Roseline; Elezzabi, Abdulhakem Y.

2017-02-01

The mechanism of femtosecond laser nanosurgical attachment is investigated in the following article. Using sub-10 femtosecond laser pulses with 800 nm central wavelength were used to attach retinoblastoma cells. During the attachment process the cell membrane phospholipid bilayers hemifuse into one shared phospholipid bilayer, at the location of attachment. Transmission electron microscopy was used in order to verify the above hypothesis. Based on the imaging results, it was concluded that the two cell membrane coalesce to form one single shared membrane. The technique of cell-cell attachment via femtosecond laser pulses could potentially serve as a platform for precise cell membrane manipulation. Manipulation of the cellular membrane is valuable for studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

2016-10-01

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Cholesterol Bilayer Domains in the Eye Lens Health: A Review.

PubMed

Widomska, Justyna; Subczynski, Witold K; Mainali, Laxman; Raguz, Marija

2017-12-01

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

Interaction of Impulsive Pressures of Cavitation Bubbles with Cell Membranes during Sonoporation

NASA Astrophysics Data System (ADS)

Kodama, Tetsuya; Koshiyama, Ken-ichiro; Tomita, Yukio; Suzuki, Maiko; Yano, Takeru; Fujikawa, Shigeo

2006-05-01

Ultrasound contrast agents (UCAs), are capable of enhancing non-invasive cytoplasmic molecular delivery in the presence of ultrasound. Collapse of UCAs may generate nano-scale cavitation bubbles, resulting in the transient permeabilization of the cell membrane. In the present study, we investigated the interaction of a cavitation bubble-induced shock wave with a cell membrane using acoustic theory and molecular dynamics (MD) simulation. From the theory, we obtained the shock wave propagation distance from the center of a cavitation bubble that would induce membrane damage. The MD simulation determined the relationship between the uptake of water molecules into the lipid bilayer and the shock wave. The interaction of the shock wave induced a structural change of the bilayer and subsequently increased the fluidity of each molecule. These changes in the bilayer due to shock waves may be an important factor in the use of UCAs to produce the transient membrane permeability during sonoporation.

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer.

PubMed

Zimmermann, Kerstin; Eells, Rebecca; Heinrich, Frank; Rintoul, Stefanie; Josey, Brian; Shekhar, Prabhanshu; Lösche, Mathias; Stern, Lawrence J

2017-10-27

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ cyt ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ cyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ cyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ cyt to the bilayers in dynamic equilibrium: one in which ζ cyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ cyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

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

PubMed

Li, He; Lykotrafitis, George

2014-08-05

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 not impose a complete attachment of the filaments to the lipid bilayer. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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 not impose a complete attachment of the filaments to the lipid bilayer. PMID:25099803

Investigating Hydrophilic Pores in Model Lipid Bilayers using Molecular Simulations: Correlating Bilayer Properties with Pore Formation Thermodynamics

PubMed Central

Hu, Yuan; Sinha, Sudipta Kumar

2015-01-01

Cell-penetrating and antimicrobial peptides show 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 pore-like membrane configurations during the solute transfer process into vesicles and cells. The initiation and formation of pores are associated with a non-trivial free energy cost, thus necessitating consideration of the factors associated with pore formation and attendant free energetics. Due to experimental and modeling challenges related to the long timescales 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 head-groups, charge states, acyl chain lengths and saturation. We probe the dependence of pore-formation barriers on area per lipid, lipid bilayer thickness, 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. Pore formation free energy is higher in peptide-lipid systems relative to the peptide-free lipid systems due to penalties to maintain solvation of charged hydrophilic solutes within the membrane environment. PMID:25614183

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.

Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

PubMed

Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

2014-03-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

PubMed Central

Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

2014-01-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

Effects of dimethyl sulfoxide in cholesterol-containing lipid membranes: a comparative study of experiments in silico and with cells.

PubMed

de Ménorval, Marie-Amélie; Mir, Lluis M; Fernández, M Laura; Reigada, Ramon

2012-01-01

Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca(2+)) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations.

Effects of Dimethyl Sulfoxide in Cholesterol-Containing Lipid Membranes: A Comparative Study of Experiments In Silico and with Cells

PubMed Central

de Ménorval, Marie-Amélie; Mir, Lluis M.; Fernández, M. Laura; Reigada, Ramon

2012-01-01

Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca2+) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations. PMID:22848583

Asymmetric distribution of charged lipids between the leaflets of a vesicle bilayer induced by melittin and alamethicin

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 dimyristoylmore » 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.« less

Interaction of PLGA and trimethyl chitosan modified PLGA nanoparticles with mixed anionic/zwitterionic phospholipid bilayers studied using molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Novak, Brian; Astete, Carlos; Sabliov, Cristina; Moldovan, Dorel

2012-02-01

Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer. Nanoparticles of PLGA are commonly used for drug delivery applications. The interaction of the nanoparticles with the cell membrane may influence the rate of their uptake by cells. Both PLGA and cell membranes are negatively charged, so adding positively charged polymers such as trimethyl chitosan (TMC) which adheres to the PLGA particles improves their cellular uptake. The interaction of 3 nm PLGA and TMC-modified-PLGA nanoparticles with lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine lipids was studied using molecular dynamics simulations. The free energy profiles as function of nanoparticles position along the normal direction to the bilayers were calculated, the distribution of phosphatidylserine lipids as a function of distance of the particle from the bilayer was calculated, and the time scale for particle motion in the directions parallel to the bilayer surface was estimated.

Bilayer membrane interactions with nanofabricated scaffolds

DOE PAGES

Collier, C. Patrick

2015-07-29

Membrane function is facilitated by lateral organization within the lipid bilayer, including phase-separation of lipids into more ordered domains (lipid rafts) and anchoring of the membrane to a cytoskeleton. These features have proven difficult to reproduce in model membrane systems such as black lipid membranes, unilamellar vesicles and supported bilayers. However, advances in micro/nanofabrication have resulted in more realistic synthetic models of membrane-cytoskeleton interactions that can help uncover the design rules responsible for biological membrane formation and organization. This review will focus on describing micro-/nanostructured scaffolds that can emulate the connections of a cellular membrane to an underlying “cytoskeleton”. Thismore » includes molecular-based scaffolds anchored to a solid substrate through surface chemistry, solid-state supports modified by material deposition, lithography and etching, the creation of micro/nanoporous arrays, integration with microfluidics, and droplet-based bilayers at interfaces. Lastly, model systems such as these are increasing our understanding of structure and organization in cell membranes, and how they result in the emergence of functionality at the nanoscale.« less

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors.

PubMed

Prior, Stephen H; Fulcher, Yan G; Koppisetti, Rama K; Jurkevich, Alexander; Van Doren, Steven R

2015-11-03

Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. Nuclear magnetic resonance structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward toward the enzyme's positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen. Copyright © 2015 Elsevier Ltd. All rights reserved.

Formation of supported lipid bilayers containing phase-segregated domains and their interaction with gold nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melby, Eric S.; Mensch, Arielle C.; Lohse, Samuel E.

2016-01-01

The cell membrane represents an important biological interface that nanoparticles may encounter after being released into the environment. Interaction of nanoparticles with cellular membranes may alter membrane structure and function, lead to their uptake into cells, and elicit adverse biological responses. Supported lipid bilayers have proven to be valuable ex vivo models for biological membranes, allowing investigation of their mechanisms of interaction with nanoparticles with a degree of control impossible in living cells. To date, the majority of research on nanoparticle interaction with supported lipid bilayers has employed membranes composed of single or binary mixtures of phospholipids. Cellular membranes containmore » a wide variety of lipids and exhibit lateral organization. Ordered membrane domains enriched in specific membrane components are referred to as lipid rafts and have not been explored with respect to their interaction with nanoparticles. Here we develop model lipid raft-containing membranes amenable to investigation by a variety of surface-sensitive analytical techniques and demonstrate that lipid rafts influence the extent of nanoparticle attachment to model membranes. We determined conditions that allow reliable formation of bilayers containing rafts enriched in sphingomyelin and cholesterol and confirmed their morphology by structured illumination and atomic force microscopies. We demonstrate that lipid rafts increase attachment of cationic gold nanoparticles to model membranes under near physiological ionic strength conditions (0.1 M NaCl) at pH 7.4. We anticipate that these results will serve as the foundation for and motivate further study of nanoparticle interaction with compositionally varied lipid rafts.« less

Depletion with Cyclodextrin Reveals Two Populations of Cholesterol in Model Lipid Membranes

PubMed Central

Litz, Jonathan P.; Thakkar, Niket; Portet, Thomas; Keller, Sarah L.

2016-01-01

Recent results provide evidence that cholesterol is highly accessible for removal from both cell and model membranes above a threshold concentration that varies with membrane composition. Here we measured the rate at which methyl-β-cyclodextrin depletes cholesterol from a supported lipid bilayer as a function of cholesterol mole fraction. We formed supported bilayers from two-component mixtures of cholesterol and a PC (phosphatidylcholine) lipid, and we directly visualized the rate of decrease in area of the bilayers with fluorescence microscopy. Our technique yields the accessibility of cholesterol over a wide range of concentrations (30–66 mol %) for many individual bilayers, enabling fast acquisition of replicate data. We found that the bilayers contain two populations of cholesterol, one with low surface accessibility and the other with high accessibility. A larger fraction of the total membrane cholesterol appears in the more accessible population when the acyl chains of the PC-lipid tails are more unsaturated. Our findings are most consistent with the predictions of the condensed-complex and cholesterol bilayer domain models of cholesterol-phospholipid interactions in lipid membranes. PMID:26840728

All-Atom Molecular Dynamics-Based Analysis of Membrane-Stabilizing Copolymer Interactions with Lipid Bilayers Probed under Constant Surface Tensions.

PubMed

Houang, Evelyne M; Bates, Frank S; Sham, Yuk Y; Metzger, Joseph M

2017-11-30

An all-atom phospholipid bilayer and triblock copolymer model was developed for molecular dynamics (MD) studies. These were performed to investigate the mechanism of interaction between membrane-stabilizing triblock copolymer P188 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers under applied lateral surface tension (γ) to model membrane mechanical stress. Results showed that P188 insertion is driven by the hydrophobic poly(propylene oxide) (PPO) core and dependent on bilayer area per lipid. Moreover, insertion of P188 increased the bilayer's resistance to mechanical rupture, as observed by a significant increase in the absolute lateral pressure required to disrupt the bilayer. To further investigate the specific chemical features of P188 underlying membrane stabilizer function, a series of MD simulations with triblock copolymers of the same class as P188 but of varying chemical composition and sizes were performed. Results showed that triblock copolymer insertion into the lipid bilayer is dependent on overall copolymer hydrophobicity, with higher copolymer hydrophobicity requiring a reduced bilayer area per lipid ratio for insertion. Further analysis revealed that the effect of copolymer insertion on membrane mechanical integrity was also dependent on hydrophobicity. Here, P188 insertion significantly increased the absolute apparent lateral pressure required to rupture the POPC bilayer, thereby protecting the membrane against mechanical stress. In marked contrast, highly hydrophobic copolymers decreased the lateral pressure necessary for membrane rupture and thus rendering the membrane significantly more susceptible to mechanical stress. These new in silico findings align with recent experimental findings using synthetic lipid bilayers and in muscle cells in vitro and mouse models in vivo. Collectively, these data underscore the importance of PEO-PPO-PEO copolymer chemical composition in copolymer-based muscle membrane stabilization in vitro and in vivo. All-atom modeling with MD simulations holds promise for investigating novel copolymers with enhanced membrane interacting properties.

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers

DOE PAGES

Carrillo, Jan-Michael Y.; Katsaras, John; Sumpter, Bobby G.; ...

2017-01-12

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to signficant insights into vital membrane phenomena such as domain formation, passive permeation and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatially and temporally. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular under- standing of lipid membranes. Particularly, the synergymore » between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. We also describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidity. Specfically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for investigations of more complex membrane systems.« less

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

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

PubMed

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

2016-11-01

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 and 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. The results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantifying the deformation of the red blood cell skeleton in shear flow

NASA Astrophysics Data System (ADS)

Peng, Zhangli; Zhu, Qiang

2012-02-01

To quantitatively predict the response of red blood cell (RBC) membrane in shear flow, we carried out multiphysics simulations by coupling a three-level multiscale approach of RBC membranes with a Boundary Element Method (BEM) for surrounding flows. Our multiscale approach includes a model of spectrins with the domain unfolding feature, a molecular-based model of the junctional complex with detailed protein connectivity and a whole cell Finite Element Method (FEM) model with the bilayer-skeleton friction derived from measured transmembrane protein diffusivity based on the Einstein-Stokes relation. Applying this approach, we investigated the bilayer-skeleton slip and skeleton deformation of healthy RBCs and RBCs with hereditary spherocytosis anemia during tank-treading motion. Compared with healthy cells, cells with hereditary spherocytosis anemia sustain much larger skeleton-bilayer slip and area deformation of the skeleton due to deficiency of transmembrane proteins. This leads to extremely low skeleton density and large bilayer-skeleton interaction force, both of which may cause bilayer loss. This finding suggests a possible mechanism of the development of hereditary spherocytosis anemia.

Study of Raft Domains in Model Membrane of DPPC/PE/Cholesterol

NASA Astrophysics Data System (ADS)

Lor, Chai; Hirst, Linda

2010-10-01

Raft domains in bilayer membrane are thought to play an important role in many cell functions such as cell signaling or trans-membrane protein activation. Here we use a model membrane consisting of DPPC/PE/cholesterol to examine the structure of membrane rafts and phase interactions. In particular we are interested in lipids containing the highly polyunsaturated fatty acid DHA. We use both atomic force microscopy (AFM) and fluorescence microscopy to obtain information on the structural properties of raft regions and track cholesterol. As expected, we find phase separation of raft regions between saturated and unsaturated lipids. Moreover, we find that the roughness of the domains change with varying cholesterol concentration possibly due to overpacking. This model study provides further understanding of the role of cholesterol in bilayer membrane leading towards a better knowledge of cell membranes.

Thylakoid membrane landscape in the sixties: a tribute to Andrew Benson.

PubMed

Anderson, Jan M

2007-05-01

Prior to the 1960s, the model for the molecular structure of cell membranes consisted of a lipid bilayer held in place by a thin film of electrostatically-associated protein stretched over the bilayer surface: (the Danielli-Davson-Robertson "unit membrane" model). Andrew Benson, an expert in the lipids of chloroplast thylakoid membranes, questioned the relevance of the unit membrane model for biological membranes, especially for thylakoid membranes, instead of emphasizing evidence in favour of hydrophobic interactions of membrane lipids within complementary hydrophobic regions of membrane-spanning proteins. With Elliot Weier, Benson postulated a remarkable subunit lipoprotein monolayer model for thylakoids. Following the advent of freeze fracture microscopy and the fluid lipid-protein mosaic model by Singer and Nicolson, the subunits, membrane-spanning integral proteins, span a dynamic lipid bilayer. Now that high resolution X-ray structures of photosystems I and II are being revealed, the seminal contribution of Andrew Benson can be appreciated.

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes.

PubMed

Suwalsky, Mario; Belmar, Jessica; Villena, Fernando; Gallardo, María José; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2013-11-01

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures. Copyright © 2013 Elsevier Inc. All rights reserved.

Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

PubMed

Richards, Mark J; Daniel, Susan

2017-02-07

The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

Characterization of Interactions between Curcumin and Different Types of Lipid Bilayers by Molecular Dynamics Simulation.

PubMed

Lyu, Yuan; Xiang, Ning; Mondal, Jagannath; Zhu, Xiao; Narsimhan, Ganesan

2018-03-01

Curcumin (CUR) is a natural food ingredient with known ability to target microbial cell membrane. In this study, the interactions of CUR with different types of model lipid bilayers (POPE, POPG, POPC, DOPC, and DPPE), mixtures of model lipid bilayers (POPE/POPG), and biological membrane mimics (Escherichia coli and yeast) were investigated by all-atom explicit solvent molecular dynamics (MD) simulation. CUR readily inserts into different types of model lipid bilayer systems in the liquid crystalline state, staying in the lipid tails region near the interface of lipid head and lipid tail. Parallel orientation to the membrane surface is found to be more probable than perpendicular for CUR, as indicated by the tilt angle distribution. This orientation preference is less significant as the fraction of POPE is increased in the system, likely due to the better water solvation of perpendicular orientation in the POPE bilayer. In E. coli and yeast bilayers, tilt angle distributions were similar to that for POPE/POPG mixed bilayer, with water hydration number around CUR for the former being higher. Insertion of CUR resulted in membrane thinning. The results from these simulations provide insights into the possible differences in membrane disrupting activity of CUR against different types of microorganisms.

Raman Imaging in Cell Membranes, Lipid-Rich Organelles, and Lipid Bilayers.

PubMed

Syed, Aleem; Smith, Emily A

2017-06-12

Raman-based optical imaging is a promising analytical tool for noninvasive, label-free chemical imaging of lipid bilayers and cellular membranes. Imaging using spontaneous Raman scattering suffers from a low intensity that hinders its use in some cellular applications. However, developments in coherent Raman imaging, surface-enhanced Raman imaging, and tip-enhanced Raman imaging have enabled video-rate imaging, excellent detection limits, and nanometer spatial resolution, respectively. After a brief introduction to these commonly used Raman imaging techniques for cell membrane studies, this review discusses selected applications of these modalities for chemical imaging of membrane proteins and lipids. Finally, recent developments in chemical tags for Raman imaging and their applications in the analysis of selected cell membrane components are summarized. Ongoing developments toward improving the temporal and spatial resolution of Raman imaging and small-molecule tags with strong Raman scattering cross sections continue to expand the utility of Raman imaging for diverse cell membrane studies.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

PubMed

Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

2013-05-28

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

PubMed

Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

2009-08-13

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

Molecular design and MD simulations of epitaxial superlattice of self-assembling ternary lipid bilayers

NASA Astrophysics Data System (ADS)

Chou, George; Vaughn, Mark; Cheng, K.

2011-10-01

Multicomponent lipid bilayers represent an important model system for studying cell membranes. At present, an ordered multicomponent phospholipid/cholesterol bilayer system involving charged lipid is still not available. Using a lipid superlattice (SL) model, a 13 x 15 x 15 nm^3 ternary phosphatidylcholine/phosphatidylserine/cholesterol bilayer system in water with simultaneous headgroup SL and acyl chain SL at different depths, or epitaxial SL, of the bilayer has been designed with atomistic detail. The arrangements of this epitaxial SL system were optimized by only two molecular parameters, lattice space and rotational angle of the lipids. Using atomistic MD simulations, we demonstrated the stability of the ordered structures for more than 100 ns. A positional restrained system was also used as a control. This system will provide new insights into understanding the nanodomain structures of cell membranes at the molecular level.

Cationic cell-penetrating peptide binds to planar lipid bilayers containing negatively charged lipids but does not induce conductive pores.

PubMed

Gurnev, Philip A; Yang, Sung-Tae; Melikov, Kamran C; Chernomordik, Leonid V; Bezrukov, Sergey M

2013-05-07

Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 μM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 μM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The insertion and transport of anandamide in synthetic lipid membranes are both cholesterol-dependent.

PubMed

Di Pasquale, Eric; Chahinian, Henri; Sanchez, Patrick; Fantini, Jacques

2009-01-01

Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide. Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide. Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide.

Probing Lipid Bilayers under Ionic Imbalance.

PubMed

Lin, Jiaqi; Alexander-Katz, Alfredo

2016-12-06

Biological membranes are normally under a resting transmembrane potential (TMP), which originates from the ionic imbalance between extracellular fluids and cytosols, and serves as electric power storage for cells. In cell electroporation, the ionic imbalance builds up a high TMP, resulting in the poration of cell membranes. However, the relationship between ionic imbalance and TMP is not clearly understood, and little is known about the effect of ionic imbalance on the structure and dynamics of biological membranes. In this study, we used coarse-grained molecular dynamics to characterize a dipalmitoylphosphatidylcholine bilayer system under ionic imbalances ranging from 0 to ∼0.06 e charges per lipid (e/Lip). We found that the TMP displayed three distinct regimes: 1) a linear regime between 0 and 0.045 e/Lip, where the TMP increased linearly with ionic imbalance; 2) a yielding regime between ∼0.045 and 0.060 e/Lip, where the TMP displayed a plateau; and 3) a poration regime above ∼0.060 e/Lip, where we observed pore formation within the sampling time (80 ns). We found no structural changes in the linear regime, apart from a nonlinear increase in the area per lipid, whereas in the yielding regime the bilayer exhibited substantial thinning, leading to an excess of water and Na + within the bilayer, as well as significant misalignment of the lipid tails. In the poration regime, lipid molecules diffused slightly faster. We also found that the fluid-to-gel phase transition temperature of the bilayer dropped below the normal value with increased ionic imbalances. Our results show that a high ionic imbalance can substantially alter the essential properties of the bilayer, making the bilayer more fluid like, or conversely, depolarization of a cell could in principle lead to membrane stiffening. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Inducing morphological changes in lipid bilayer membranes with microfabricated substrates

NASA Astrophysics Data System (ADS)

Liu, Fangjie; Collins, Liam F.; Ashkar, Rana; Heberle, Frederick A.; Srijanto, Bernadeta R.; Collier, C. Patrick

2016-11-01

Lateral organization of lipids and proteins into distinct domains and anchoring to a cytoskeleton are two important strategies employed by biological membranes to carry out many cellular functions. However, these interactions are difficult to emulate with model systems. Here we use the physical architecture of substrates consisting of arrays of micropillars to systematically control the behavior of supported lipid bilayers - an important step in engineering model lipid membrane systems with well-defined functionalities. Competition between attractive interactions of supported lipid bilayers with the underlying substrate versus the energy cost associated with membrane bending at pillar edges can be systematically investigated as functions of pillar height and pitch, chemical functionalization of the microstructured substrate, and the type of unilamellar vesicles used for assembling the supported bilayer. Confocal fluorescent imaging and AFM measurements highlight correlations that exist between topological and mechanical properties of lipid bilayers and lateral lipid mobility in these confined environments. This study provides a baseline for future investigations into lipid domain reorganization on structured solid surfaces and scaffolds for cell growth.

Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Choubey, Amit

Biological cell membranes provide mechanical stability to cells and understanding their structure, dynamics and mechanics are important biophysics problems. Experiments coupled with computational methods such as molecular dynamics (MD) have provided insight into the physics of membranes. We use long-time and large-scale MD simulations to study the structure, dynamics and mechanical behavior of membranes. We investigate shock-induced collapse of nanobubbles in water using MD simulations based on a reactive force field. We observe a focused jet at the onset of bubble shrinkage and a secondary shock wave upon bubble collapse. The jet length scales linearly with the nanobubble radius, as observed in experiments on micron-to-millimeter size bubbles. Shock induces dramatic structural changes, including an ice-VII-like structural motif at a particle velocity of 1 km/s. The incipient ice VII formation and the calculated Hugoniot curve are in good agreement with experimental results. We also investigate molecular mechanisms of poration in lipid bilayers due to shock-induced collapse of nanobubbles. Our multimillion-atom MD simulations reveal that the jet impact generates shear flow of water on bilayer leaflets and pressure gradients across them. This transiently enhances the bilayer permeability by creating nanopores through which water molecules translocate rapidly across the bilayer. Effects of nanobubble size and temperature on the porosity of lipid bilayers are examined. The second research project focuses on cholesterol (CHOL) dynamics in phospholipid bilayers. Several experimental and computational studies have been performed on lipid bilayers consisting of dipalmitoylphosphatidylcholine (DPPC) and CHOL molecules. CHOL interleaflet transport (flip-flop) plays an important role in interleaflet coupling and determining CHOL flip-flop rate has been elusive. Various studies report that the rate ranges between milliseconds to seconds. We calculate CHOL flip-flop rates by performing a 15 mus all-atom MD simulation of a DPPC-CHOL bilayer. We find that the CHOL flip-flop rates are on the sub microsecond timescale. These results are verified by performing various independent parallel replica (PR) simulations. Our PR simulations provide significant boost in sampling of the flip-flop events. We observe that the CHOL flip-flop can induce membrane order, regulate membrane-bending energy, and facilitate membrane relaxation. The rapid flip-flop rates reported here have important implications for the role of CHOL in mechanical properties of cell membranes, formation of domains, and maintaining CHOL concentration asymmetry in plasma membrane. Our PR approach can reach submillisecond time scales and bridge the gap between MD simulations and Nuclear Magnetic Resonance (NMR) experiments on CHOL flip-flop dynamics in membranes. The last project deals with transfection barriers encountered by a bare small interfering RNA (siRNA) in a phospholipid bilayer. SiRNA molecules play a pivotal role in therapeutic applications. A key limitation to the widespread implementation of siRNA-based therapeutics is the difficulty of delivering siRNA-based drugs to cells. We have examined structural and mechanical barriers to siRNA passage across a phospholipid bilayer using all-atom MD simulations. We find that the electrostatic interaction between the anionic siRNA and head groups of phospholipid molecules induces a phase transformation from the liquid crystalline to ripple phase. Steered MD simulations reveal that the siRNA transfection through the ripple phase requires a force of ˜ 1.5 nN.

Aromaticity/Bulkiness of Surface Ligands to Promote the Interaction of Anionic Amphiphilic Gold Nanoparticles with Lipid Bilayers.

PubMed

Gao, Jinhong; Zhang, Ouyang; Ren, Jing; Wu, Chuanliu; Zhao, Yibing

2016-02-16

The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity.

Engineering plant membranes using droplet interface bilayers.

PubMed

Barlow, N E; Smpokou, E; Friddin, M S; Macey, R; Gould, I R; Turnbull, C; Flemming, A J; Brooks, N J; Ces, O; Barter, L M C

2017-03-01

Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana , tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.

Dynamic patterns in a supported lipid bilayer driven by standing surface acoustic waves.

PubMed

Hennig, Martin; Neumann, Jürgen; Wixforth, Achim; Rädler, Joachim O; Schneider, Matthias F

2009-11-07

In the past decades supported lipid bilayers (SLBs) have been an important tool in order to study the physical properties of biological membranes and cells. So far, controlled manipulation of SLBs is very limited. Here we present a new technology to create lateral patterns in lipid membranes controllable in both space and time. Surface acoustic waves (SAWs) are used to generate lateral standing waves on a piezoelectric substrate which create local "traps" in the lipid bilayer and lead to a lateral modulation in lipid concentration. We demonstrate that pattern formation is reversible and does not affect the integrity of the lipid bilayer as shown by extracting the diffusion constant of fluid membranes. The described method could possibly be used to design switchable interfaces for the lateral transport and organization of membrane bound macromolecules to create dynamic bioarrays and control biofilm formation.

Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

NASA Astrophysics Data System (ADS)

Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

2016-09-01

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

PubMed

Plesnar, Elzbieta; Szczelina, Robert; Subczynski, Witold K; Pasenkiewicz-Gierula, Marta

2018-02-01

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (P M ) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the P M across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the P M of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer

DOE PAGES

Taylor, Graham J.; Venkatesan, Guru A.; Collier, C. Patrick; ...

2015-08-05

In this study, thickness and tension are important physical parameters of model cell membranes. However, traditional methods to measure these quantities require multiple experiments using separate equipment. This work introduces a new multi-step procedure for directly accessing in situ multiple physical properties of droplet interface bilayers (DIB), including specific capacitance (related to thickness), lipid monolayer tension in the Plateau-Gibbs border, and bilayer tension. The procedure employs a combination of mechanical manipulation of bilayer area followed by electrowetting of the capacitive interface to examine the sensitivities of bilayer capacitance to area and contact angle to voltage, respectively. These data allow formore » determining the specific capacitance of the membrane and surface tension of the lipid monolayer, which are then used to compute bilayer thickness and tension, respectively. The use of DIBs affords accurate optical imaging of the connected droplets in addition to electrical measurements of bilayer capacitance, and it allows for reversibly varying bilayer area. After validating the accuracy of the technique with diphytanoyl phosphatidylcholine (DPhPC) DIBs in hexadecane, the method is applied herein to quantify separately the effects on membrane thickness and tension caused by varying the solvent in which the DIB is formed and introducing cholesterol into the bilayer. Because the technique relies only on capacitance measurements and optical images to determine both thickness and tension, this approach is specifically well-suited for studying the effects of peptides, biomolecules, natural and synthetic nanoparticles, and other species that accumulate within membranes without altering bilayer conductance.« less

Lipid bilayers suspended on microfabricated supports

NASA Astrophysics Data System (ADS)

Ogier, Simon D.; Bushby, Richard J.; Cheng, Yaling; Cox, Tim I.; Evans, Stephen D.; Knowles, Peter F.; Miles, Robert E.; Pattison, Ian

2001-03-01

The plasma membrane, that exists as part of many animal and plant cells, is a regulator for the transport of ions and small molecules across cell boundaries. Two main components involved are the phospholipid bilayer and the transport proteins. This paper details the construction of a micromachined support for bilayers (MSB) as a first step towards the development of highly selective and highly sensitive ion-channel based biosensors. The device consists of a ~100 micrometer hole in a polymeric support above a cavity that can hold ~25 nL of electrolyte. Electrodes attached to the structure allow the resistance of the membranes to be measured using d.c. conductivity. The MSB is made in two halves, using SU8 ultra-thick resist, which are subsequently bonded together to make the final structure. A layer of gold, surrounding the aperture, enables self-assembled monolayers of alkanethiols to be used to make the polymeric structure biocompatible. Lipid membranes have been formed over these holes with resistances comparable with those of natural membranes >10 MOhmcm^2. The ion-channel gramicidin has successfully been incorporated into the bilayer and its activity monitored. It is proposed that this type of device could be used not only for studying membrane transport phenomena but also as part of an ion-channel based biosensor.

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

PubMed Central

Grover, Rahul; Fischer, Janine; Schwarz, Friedrich W.; Walter, Wilhelm J.; Schwille, Petra; Diez, Stefan

2016-01-01

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. PMID:27803325

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

DOE PAGES

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc; ...

2017-06-13

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less

Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment.

PubMed

Sani, Marc-Antoine; Gagne, Eve; Gehman, John D; Whitwell, Thomas C; Separovic, Frances

2014-09-01

A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.

Novel bilayer wound dressing based on electrospun gelatin/keratin nanofibrous mats for skin wound repair.

PubMed

Yao, Chun-Hsu; Lee, Chia-Yu; Huang, Chiung-Hua; Chen, Yueh-Sheng; Chen, Kuo-Yu

2017-10-01

A bilayer membrane (GKU) with a commercial polyurethane wound dressing as an outer layer and electrospun gelatin/keratin nanofibrous mat as an inner layer was fabricated as a novel wound dressing. Scanning electron micrographs showed that gelatin/keratin nanofibers had a uniform morphology and bead-free structure with average fiber diameter of 160.4nm. 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay using L929 fibroblast cells indicated that the residues released from the gelatin/keratin composite nanofibrous mat accelerated cell proliferation. Cell attachment experiments revealed that adhered cells spread better and migrated deeper into the gelatin/keratin nanofibrous mat than that into the gelatin nanofibrous mat. In animal studies, compared with the bilayer membrane without keratin, gauze and commercial wound dressing, Comfeel®, GKU membrane gave much more number of blood vessels and a greater reduction in wound area at 4days, and better wound repair at 14days with a thicker epidermis and larger number of newly formed hair follicles. GKU membrane, thus, could be a good candidate for wound dressing applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of Transmembrane Potential and Defects on the Permeabilization of Lipid Bilayers by Alamethicin, an Ion-Channel-Forming Peptide.

PubMed

Su, ZhangFei; Shodiev, Muzaffar; Leitch, J Jay; Abbasi, Fatemeh; Lipkowski, Jacek

2018-05-29

The insertion and ion-conducting channel properties of alamethicin reconstituted into a 1,2-di- O-phytanyl- sn-glycero-3-phosphocholine bilayer floating on the surface of a gold (111) electrode modified with a 1-thio-β-d-glucose (β-Tg) self-assembled monolayer were investigated using a combination of electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The hydrophilic β-Tg monolayer separated the bilayer from the gold substrate and created a water-rich spacer region, which better represents natural cell membranes. The EIS measurements acquired information about the membrane resistivity (a measure of membrane porosity), and the PM-IRRAS experiments provided insight into the conformation and orientation of the membrane constituents as a function of the transmembrane potential. The results showed that the presence of alamethicin had a small effect on the conformation and orientation of phospholipid molecules within the bilayer for all studied potentials. In contrast, the alamethicin peptides assumed a surface state, where the helical axes adopted a large tilt angle with respect to the surface normal, at small transmembrane potentials, and inserted into the bilayer at sufficiently negative transmembrane potentials forming pores, which behaved as barrel-stave ion channels for ionic transport across the membrane. The results indicated that insertion of alamethincin peptides into the bilayer was driven by the dipole-field interactions and that the transitions between the inserted and surface states were electrochemically reversible. Additionally, the EIS measurements performed on phospholipid bilayers without alamethicin also showed that the application of negative transmembrane potentials introduces defects into the bilayer. The membrane resistances measured in both the absence and presence of alamethicin show similar dependencies on the electrode potential, suggesting that the insertion of the peptide may also be assisted by the electroporation of the membrane. The findings in this study provide new insights into the mechanism of alamethicin insertion into phospholipid bilayers.

Loss of electrostatic cell-surface repulsion mediates myelin membrane adhesion and compaction in the central nervous system.

PubMed

Bakhti, Mostafa; Snaidero, Nicolas; Schneider, David; Aggarwal, Shweta; Möbius, Wiebke; Janshoff, Andreas; Eckhardt, Matthias; Nave, Klaus-Armin; Simons, Mikael

2013-02-19

During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.

Effects of fullerene on lipid bilayers displaying different liquid ordering: a coarse-grained molecular dynamics study.

PubMed

Sastre, Judit; Mannelli, Ilaria; Reigada, Ramon

2017-11-01

The toxic effects and environmental impact of nanomaterials, and in particular of Fullerene particles, are matters of serious concern. It has been reported that fullerene molecules enter the cell membrane and occupy its hydrophobic region. Understanding the effects of carbon-based nanoparticles on biological membranes is therefore of critical importance to determine their exposure risks. We report on a systematic coarse-grained molecular dynamics study of the interaction of fullerene molecules with simple model cell membranes. We have analyzed bilayers consisting of lipid species with different degrees of unsaturation and a variety of cholesterol fractions. Addition of fullerene particles to phase-segregated ternary membranes is also investigated in the context of the lipid raft model for the organization of the cell membrane. Fullerene addition to lipid membranes modifies their structural properties like thickness, area and internal ordering of the lipid species, as well as dynamical aspects such as molecular diffusion and cholesterol flip-flop. Interestingly, we show that phase-segregating ternary lipid membranes accumulate fullerene molecules preferentially in the liquid-disordered domains promoting phase-segregation and domain alignment across the membrane. Lipid membrane internal ordering determines the behavior and distribution of fullerene particle, and this, in turn, determines the influence of fullerene on the membrane. Lipid membranes are good solvents of fullerene molecules, and in particular those with low internal ordering. Preference of fullerene molecules to be dissolved in the more disordered hydrophobic regions of a lipid bilayer and the consequent alteration of its phase behavior may have important consequences on the activity of biological cell membranes and on the bioconcentration of fullerene in living organisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Modeling the Soft Geometry of Biological Membranes

NASA Astrophysics Data System (ADS)

Daly, K.

This dissertation presents work done applying the techniques of physics to biological systems. The difference in length scales of the thickness of the phospolipid bilayer and overall size of a biological cell allows bilayer to be modeled elastically as a thin sheet. The Helfrich free energy is extended applied to models representing various biological systems, in order to find quasi-equilibrium states as well as transitions between states. Morphologies are approximated as axially sym-metric. Stable morphologies are de-termined analytically and through the use of computer simulation. The simple morphologies examined analytically give a model for the pearling transition seen in growing biological cells. An analytic model of celluar bulging in gram-negative bacteria predicts a critical pore radius for bulging of 20 nanometers. This model is extended to the membrane dynamics of human red blood cells, predicting three morphologic phases which are seen in vivo. A computer simulation was developed to study more complex morphologies with models representing different bilayer compositions. Single and multi-component bilayer models reproduce morphologies previously predicted by Seifert. A mean field model representing the intrinsic curvature of proteins coupling to membrane curvature is used to explore the stability of the particular morphology of rod outer segment cells. The process of pore formation and expansion in cell-cell fusion is not well understood. Simulation of the pore created in cell-cell fusion led to the finding of a minimal pore radius required for pore expansion, suggesting pores formed in nature are formed with a minimum size.

The Lipid domain Phase diagram in a Dipalmitoyl-PC/Docosahaexnoic Acid-PE/Cholesterol System

NASA Astrophysics Data System (ADS)

Lor, Chai; Hirst, Linda

2011-03-01

Lipid domains in bilayer membrane and polyunsaturated fatty acids (PUFAs) are thought to play an important role in cellular activities. In particular, lipids containing docosahaexnoic acid are an interesting class of PUFAs due to their health benefits. In this project, we perform oxidation measurements of DHA-PE to determine the rate of oxidation in combination with antioxidants. A ternary diagram of DPPC/DHA-PE/cholesterol is mapped out to identify phase separation phenomena using atomic force microscope (AFM). Fluorescence microscopy is also used to image lipid domains in a flat bilayer with fluorescent labels. As expected, we observe the phase, shape, and size of lipid domains changes with varying composition. Moreover, we find that the roughness of the domains changes possibly due to overpacking of cholesterol in domains. This model study provides further understanding of the role of cholesterol in the bilayer membrane leading towards a better understanding of cell membranes. NSF award # DMR 0852791, ``CAREER: Self-Assembly of Polyunsaturated Lipids and Cholesterol In The Cell Membrane.''

The Effects of Polyunsaturated Lipid Components on bilayer Structure

NASA Astrophysics Data System (ADS)

Pramudya, Y.; Kiss, A.; Nguyen, Lam T.; Yuan, J.; Hirst, Linda S.

2007-03-01

Polyunsaturated fatty acids (PUFAs), such as DHA (Docosahexanoic Acid) and AA (Alphalinoleic Acid) have been the focus of much research attention in recent years, due to their apparent health benefits and effects on cell physiology. They are found in a variety of biological membranes and have been implicated with lipid raft formation and possible function, particularly in the retinal rod cells and the central nervous system. In this work lipid bilayer structure has been investigated in lipid mixtures, incorporating polyunsaturated fatty acid moieties. The structural effects of increasing concentrations of both symmetric and asymmetric PUFA materials on the bilayer structure are investigated via synchrotron x-ray diffraction on solution samples. We observe bilayer spacings to increase with the percentage of unsaturated fatty acid lipid in the membrane, whilst the degree of ordering significantly decreases. In fact above 20% of fatty acid, well defined bilayers are no longer observed to form. Evidence of phase separation can be clearly seen from these x-ray results and in combination with AFM measurements.

Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers.

PubMed

Lauridsen, Holly M; Gonzalez, Anjelica L

2017-01-01

The vascular basement membrane-a thin, elastic layer of extracellular matrix separating and encasing vascular cells-provides biological and mechanical cues to endothelial cells, pericytes, and migrating leukocytes. In contrast, experimental scaffolds typically used to replicate basement membranes are stiff and bio-inert. Here, we present thin, porated polyethylene glycol hydrogels to replicate human vascular basement membranes. Like commercial transwells, our hydrogels are approximately 10μm thick, but like basement membranes, the hydrogels presented here are elastic (E: 50-80kPa) and contain a dense network of small pores. Moreover, the inclusion of bioactive domains introduces receptor-mediated biochemical signaling. We compare elastic hydrogels to common culture substrates (E: >2GPa) for human endothelial cell and pericyte monolayers and bilayers to replicate postcapillary venules in vitro. Our data demonstrate that substrate elasticity facilitates differences in vascular phenotype, supporting expression of vascular markers that are increasingly replicative of venules. Endothelial cells differentially express vascular markers, like EphB4, and leukocyte adhesion molecules, such as ICAM-1, with decreased mechanical stiffness. With porated PEG hydrogels we demonstrate the ability to evaluate and observe leukocyte recruitment across endothelial cell and pericyte monolayers and bilayers, reporting that basement membrane scaffolds can significantly alter the rate of vascular migration in experimental systems. Overall, this study demonstrates the creation and utility of a new and accessible method to recapture the mechanical and biological complexity of human basement membranes in vitro.

Composite fuel cell membranes

DOEpatents

Plowman, K.R.; Rehg, T.J.; Davis, L.W.; Carl, W.P.; Cisar, A.J.; Eastland, C.S.

1997-08-05

A bilayer or trilayer composite ion exchange membrane is described suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

Composite fuel cell membranes

DOEpatents

Plowman, Keith R.; Rehg, Timothy J.; Davis, Larry W.; Carl, William P.; Cisar, Alan J.; Eastland, Charles S.

1997-01-01

A bilayer or trilayer composite ion exchange membrane suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

PubMed

Biswas, Nupur; Bhattacharya, Rupak; Saha, Arindam; Jana, Nikhil R; Basu, Jaydeep K

2015-10-07

Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

Studies of Water Diffusion on Single-Supported Bilayer Lipid Membranes by Quasielastic Neutron Scattering

NASA Astrophysics Data System (ADS)

Bai, M.; Miskowiec, A.; Wang, S.-K.; Taub, H.; Jenkins, T.; Tyagi, M.; Neumann, D. A.; Hansen, F. Y.

2010-03-01

Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently demonstrated the feasibility of using quasielastic neutron scattering to study on a ˜1 ns time scale the diffusion of water bound to single-supported bilayer lipid membranes. Two different membrane samples characterized by AFM were investigated: protonated DMPC + D2O and tail-deuterated DMPC + H2O. Both fully hydrated membranes were deposited onto SiO2-coated Si(100) substrates. Measurements of elastic neutron intensity as a function of temperature on the High Flux Backscattering Spectrometer at NIST reveal features in the diffusive motion of water that have not been observed previously using multilayer membrane stacks. On slow cooling, the elastic intensity shows sharp step-like increases in the temperature range 265 to 272 K that we tentatively interpret as successive mobile-to-immobile transitions of water bound to the membrane.

Interactive transport of guanidinylated poly(propylene imine)-based dendrimers through liposomal and cellular membranes.

PubMed

Tsogas, Ioannis; Sideratou, Zili; Tsiourvas, Dimitris; Theodossiou, Theodossis A; Paleos, Constantinos M

2007-10-15

The ability of guanidinylated poly(propylene imine) dendrimers to translocate across lipid bilayers was assessed by employing either a model phosphate-bearing liposomal membrane system or A549 human lung carcinoma cells. Two dendrimer generations, differing in the number of surface guanidinium groups, were employed, while surface acetylation or the use of spacers affected the binding of the guanidinium group to the phosphate moiety and finally the transport efficiency. Following adhesion of dendrimers with liposomes, fusion or transport occurred. Transport through the liposomal bilayer was observed at low guanidinium/phosphate molar ratios, and was enhanced when the bilayer was in the liquid-crystalline phase. For effective transport through the liposomal membrane, an optimum balance between the binding strength and the degree of hydrophobicity of the guanidinylated dendrimer is required. In experiments performed in vitro with cells, efficient penetration and internalization in subcellular organelles and cytosol was observed.

SFG studies on interactions between antimicrobial peptides and supported lipid bilayers.

PubMed

Chen, Xiaoyun; Chen, Zhan

2006-09-01

The mode of action of antimicrobial peptides (AMPs) in disrupting cell membrane bilayers is of fundamental importance in understanding the efficiency of different AMPs, which is crucial to design antibiotics with improved properties. Recent developments in the field of sum frequency generation (SFG) vibrational spectroscopy have made it a powerful and unique biophysical technique in investigating the interactions between AMPs and a single substrate supported planar lipid bilayer. We will review some of the recent progress in applying SFG to study membrane lipid bilayers and discuss how SFG can provide novel information such as real-time bilayer structure change and AMP orientation during AMP-lipid bilayer interactions in a very biologically relevant manner. Several examples of applying SFG to monitor such interactions between AMPs and a dipalmitoyl phosphatidylglycerol (DPPG) bilayer are presented. Different modes of actions are observed for melittin, tachyplesin I, d-magainin 2, MSI-843, and a synthetic antibacterial oligomer, demonstrating that SFG is very effective in the study of AMPs and AMP-lipid bilayer interactions.

Bilayer Effects of Antimalarial Compounds

PubMed Central

Ramsey, Nicole B.; Andersen, Olaf S.

2015-01-01

Because of the perpetual development of resistance to current therapies for malaria, the Medicines for Malaria Venture developed the Malaria Box to facilitate the drug development process. We tested the 80 most potent compounds from the box for bilayer-mediated effects on membrane protein conformational changes (a measure of likely toxicity) in a gramicidin-based stopped flow fluorescence assay. Among the Malaria Box compounds tested, four compounds altered membrane properties (p< 0.05); MMV007384 stood out as a potent bilayer-perturbing compound that is toxic in many cell-based assays, suggesting that testing for membrane perturbation could help identify toxic compounds. In any case, MMV007384 should be approached with caution, if at all. PMID:26551613

Bilayer Effects of Antimalarial Compounds.

PubMed

Ramsey, Nicole B; Andersen, Olaf S

2015-01-01

Because of the perpetual development of resistance to current therapies for malaria, the Medicines for Malaria Venture developed the Malaria Box to facilitate the drug development process. We tested the 80 most potent compounds from the box for bilayer-mediated effects on membrane protein conformational changes (a measure of likely toxicity) in a gramicidin-based stopped flow fluorescence assay. Among the Malaria Box compounds tested, four compounds altered membrane properties (p< 0.05); MMV007384 stood out as a potent bilayer-perturbing compound that is toxic in many cell-based assays, suggesting that testing for membrane perturbation could help identify toxic compounds. In any case, MMV007384 should be approached with caution, if at all.

Membrane Deformation and Permeabilization Caused by Microplasma Irradiation

NASA Astrophysics Data System (ADS)

Motomura, Hideki; Nagaiwa, Hidenori; Yamamoto, Kenta; Kido, Yugo; Ikeda, Yoshihisa; Satoh, Susumu; Jinno, Masafumi

2016-09-01

The microplasma irradiation achieves high gene taransfection efficiency and high cell survivability simultaneously. For this purpose, we have developed a special plasma source using a microcapillary electrode. However, it is not clear how the stimuli of effective factors generated by plasma, such as current, charge, field, chemical species, cause transfection. In this study, we used artificial cell which is a spherical vesicle consisting of a lipid bilayer to visualize membrane dynamics and permeabilization caused by microplasma irradiation. Dioleoyl phosphatidylcholine (DOPC) was used as phospholipid molecules forming the lipid bilayer. The artificial cells were prepared by natural swelling method. Fluorescent labeled polyethylene glycol (PEG) polymers (Nanocs, MPEG Fluorescein, MW = 1000) were encapsulated in the artificial cells. The artificial cells were exposed to the microplasma for 5 ms and 10-20% of decrease of the dye fluorescence in the artificial cells was observed. This result suggests the outflow of the MPEG polymers through temporary poration or deformation of the lipid bilayer. The membrane deformation dynamics was directly observed with a microscope and the relationship to the polymer outflow will be shown at the conference. This work was partly supported by a Grant-in-Aid (25108509 and 15H00896) from JSPS and a grant from Ehime University.

Neutron scattering to study membrane systems: from lipid vesicles to living cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nickels, Jonathan D.; Chatterjee, Sneha; Stanley, Christopher B.

The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent workmore » using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.« less

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

How Membrane-Active Peptides Get into Lipid Membranes.

PubMed

Sani, Marc-Antoine; Separovic, Frances

2016-06-21

The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure-function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide diversity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.

Advances in nanopatterned and nanostructured supported lipid membranes and their applications.

PubMed

Reimhult, Erik; Baumann, Martina; Kaufmann, Stefan; Kumar, Karthik; Spycher, Philipp

2010-01-01

Lipid membranes are versatile and convenient alternatives to study the properties of natural cell membranes. Self-assembled, artificial, substrate-supported lipid membranes have taken a central role in membrane research due to a combination of factors such as ease of creation, control over complexity, stability and the applicability of a large range of different analytical techniques. While supported lipid bilayers have been investigated for several decades, recent advances in the understanding of the assembly of such membranes from liposomes have spawned a renaissance in the field. Supported lipid bilayers are a highly promising tool to study transmembrane proteins in their native state, an application that could have tremendous impact on, e.g. drug discovery, development of biointerfaces and as platforms for glycomics and probing of multivalent binding which requires ligand mobility. Parallel advances in microfluidics, biosensor design, micro- and nanofabrication have converged to bring self-assembled supported lipid bilayers closer to a versatile and easy to use research tool as well as closer to industrial applications. The field of supported lipid bilayer research and application is thus rapidly expanding and diversifying with new platforms continuously being proposed and developed. In order to use supported lipid bilayers for such applications several advances have to be made: decoupling of the membrane from the support while maintaining it close to the surface, making use of biologically relevant lipid compositions, patterning of lipid membranes into arrays, and application to nanostructured substrates and sensors. This review summarizes recent advances in the field which addresses these challenges.

Behavior of 2,6-bis(decyloxy)naphthalene inside lipid bilayer.

PubMed

Kepczynski, Mariusz; Kumorek, Marta; Stepniewski, Michał; Róg, Tomasz; Kozik, Bartłomiej; Jamróz, Dorota; Bednar, Jan; Nowakowska, Maria

2010-12-02

Interactions between small organic molecules and lipid or cell membranes are important because of their role in the distribution of biologically active substances inside the membrane and their permeation through the cell membranes. In the current paper, we have explored the effect of the attachment of long hydrocarbon tails on the behavior of small organic molecule inside the lipid membrane. Naphthalene with two decyloxy groups attached at the opposite sites of the ring (2,6-bis(decyloxy)naphthalene, 3) was synthesized and incorporated into phosphatidylcholine (PC) vesicles. Fluorescence methods as well as molecular dynamic (MD) simulations were used to estimate the position, orientation, and migration of compound 3 in PC bilayer. It was found that the naphthalene ring of compound 3 resides in the upper acyl chain region of the bilayer and the hydrocarbon tails are directed to the center of the bilayer. As was shown with cryotransmission electron microscopy (cryo-TEM), such lipidlike conformation enables compound 3 to be incorporated into liposomes at a very high content without their disintegration. Moreover, compound 3 can migrate from one leaflet to other. The mechanism of this process is, however, different from that characteristic of the flip-flop event of lipid molecules in the membrane. Finally, the possible application of compound 3 as a rotational molecular probe for monitoring fluidity of liposomal membrane in the acyl side chain region was checked by studies of the effect of cholesterol on the fluorescence anisotropy of 3.

Continuous planar phospholipid bilayer supported on porous silicon thin film reflector.

PubMed

Cunin, Frédérique; Milhiet, Pierre-Emmanuel; Anglin, Emily; Sailor, Michael J; Espenel, Cédric; Le Grimellec, Christian; Brunel, Daniel; Devoisselle, Jean-Marie

2007-10-01

Reconstituting artificial membranes for in vitro studies of cell barrier mechanisms and properties is of major interest in biology. Here, artificial membranes supported on porous silicon photonic crystal reflectors are prepared and investigated. The materials are of interest for label-free probing of supported membrane events such as protein binding, molecular recognition, and transport. The porous silicon substrates are prepared as multilayered films consisting of a periodically varying porosity, with pore dimensions of a few nanometers in size. Planar phospholipid bilayers are deposited on the topmost surface of the oxidized hydrophilic mesoporous silicon films. Atomic force microscopy provides evidence of continuous bilayer deposition at the surface, and optical measurements indicate that the lipids do not significantly infiltrate the porous region. The presence of the supported bilayer does not obstruct the optical spectrum from the porous silicon layer, suggesting that the composite structures can act as effective optical biosensors.

Organization of Lipids in Fiber-Cell Plasma Membranes of the Eye Lens

PubMed Central

Subczynski, Witold K.; Mainali, Laxman; Raguz, Marija; O’Brien, William J.

2016-01-01

The plasma membrane together with the cytoskeleton forms the only supramolecular structure of the matured fiber cell which accounts for mostly all fiber cell lipids. The purpose of this review is to inform researchers about the importance of the lipid bilayer portion of the lens fiber cell plasma membranes in the maintaining lens homeostasis, and thus protecting against cataract development. PMID:26988627

Unraveling supported lipid bilayer formation kinetics: osmotic effects.

PubMed

Hain, Nicole; Gallego, Marta; Reviakine, Ilya

2013-02-19

Solid-supported lipid bilayers are used as cell membrane models and form the basis of biomimetic and biosensor platforms. The mechanism of their formation from adsorbed liposomes is not well-understood. Using membrane-permeable solute glycerol, impermeable solutes sucrose and dextran, and a pore forming peptide melittin, we studied experimentally how osmotic effects affect the kinetics of the adsorbed liposome-to-bilayer transition. We find that its rate is enhanced if adsorbed liposomes are made permeable but is not significantly retarded by impermeable solutes. The results are explained in terms of adsorbed liposome deformation and formation of transmembrane pores.

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.

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes.more » According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. - Highlights: • The interaction of memantine with human erythrocytes and lipid bilayers were assessed. • Memantine induced morphological changes to human erythrocytes. • Memantine interacted with classes of phospholipids present in the erythrocyte membrane. • Results support the hypothesis that memantine interacts with NMDA receptors.« less

Impact of cholesterol on voids in phospholipid membranes

NASA Astrophysics Data System (ADS)

Falck, Emma; Patra, Michael; Karttunen, Mikko; Hyvönen, Marja T.; Vattulainen, Ilpo

2004-12-01

Free volume pockets or voids are important to many biological processes in cell membranes. Free volume fluctuations are a prerequisite for diffusion of lipids and other macromolecules in lipid bilayers. Permeation of small solutes across a membrane, as well as diffusion of solutes in the membrane interior are further examples of phenomena where voids and their properties play a central role. Cholesterol has been suggested to change the structure and function of membranes by altering their free volume properties. We study the effect of cholesterol on the properties of voids in dipalmitoylphosphatidylcholine (DPPC) bilayers by means of atomistic molecular dynamics simulations. We find that an increasing cholesterol concentration reduces the total amount of free volume in a bilayer. The effect of cholesterol on individual voids is most prominent in the region where the steroid ring structures of cholesterol molecules are located. Here a growing cholesterol content reduces the number of voids, completely removing voids of the size of a cholesterol molecule. The voids also become more elongated. The broad orientational distribution of voids observed in pure DPPC is, with a 30% molar concentration of cholesterol, replaced by a distribution where orientation along the bilayer normal is favored. Our results suggest that instead of being uniformly distributed to the whole bilayer, these effects are localized to the close vicinity of cholesterol molecules.

Model for the dynamic responses of taste receptor cells to salty stimuli. I. Function of lipid bilayer membranes.

PubMed Central

Naito, M; Fuchikami, N; Sasaki, N; Kambara, T

1991-01-01

The dynamic response of the lipid bilayer membrane is studied theoretically using a microscopic model of the membrane. The time courses of membrane potential variations due to monovalent salt stimulation are calculated explicitly under various conditions. A set of equations describing the time evolution of membrane surface potential and diffusion potential is derived and solved numerically. It is shown that a rather simple membrane such as lipid bilayer has functions capable of reproducing the following properties of dynamic response observed in gustatory receptor potential. Initial transient depolarization does not occur under Ringer adaptation but does under water. It appears only for comparatively rapid flows of stimuli, the peak height of transient response is expressed by a power function of the flow rate, and the membrane potential gradually decreases after reaching its peak under long and strong stimulation. The dynamic responses in the present model arise from the differences between the time dependences in the surface potential phi s and the diffusion potential phi d across a membrane. Under salt stimulation phi d cannot immediately follow the variation in phi s because of the delay due to the charging up of membrane capacitance. It is suggested that lipid bilayer in the apical membrane is the most probable agency producing the initial phasic response to the stimulation. PMID:1873461

Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A–I and High Density Lipoproteins

PubMed Central

Sánchez, Susana A.; Tricerri, M. Alejandra; Ossato, Giulia; Gratton, Enrico

2010-01-01

Summary Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. FCS (Fluorescence Correlation Spectroscopy) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan GP (Generalized Polarization) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A–I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis, and offer a methodological design suited to different biological systems. PMID:20347719

Molecular dynamics simulations of salicylate effects on the micro- and mesoscopic properties of a dipalmitoylphosphatidylcholine bilayer†

PubMed Central

Song, Yuhua; Guallar, Victor; Baker, Nathan A.

2008-01-01

Salicylate, an amphiphilic molecule and a popular member of non-steroidal antiinflammatory drug family, is known to affect hearing through reduction of the electromechanical coupling in the outer hair cells of the ear. This reduction of electromotility by salicylate has been widely studied but the molecular mechanism of the phenomenon is still unknown. In this study, we investigated one aspect of salicylate’s action; namely, the perturbation of electrical and mechanical membrane properties by salicylate in the absence of cytoskeletal or membrane-bound motor proteins such as prestin. In particular, we simulated the interaction of salicylate with a dipalmitoylphosphatidylcholine (DPPC) bilayer via atomically-detailed molecular dynamics simulations to observe the effect of salicylate on the microscopic and mesoscopic properties of the bilayer. The results demonstrate that salicylate interacts with the bilayer by associating at the water-DPPC interface in a nearly perpendicular orientation and penetrating more deeply into the bilayer than either sodium or chloride. This association has several affects on the membrane properties. First, binding of salicylate to the membrane displaces chloride from the bilayer-water interface. Second, salicylate influences the electrostatic potential and dielectric properties of the bilayer, with significant changes at the water-lipid bilayer interface. Third, salicylate association results in structural changes including decreased head group area per lipid and increased lipid tail order. However, salicylate does not significantly alter the mechanical properties of the DPPC bilayer; bulk compressibility, area compressibility, and bending modulus were only perturbed by small, statistically-insignificant amounts, by the presence of salicylate. The observations from these simulations are in qualitative agreement with experimental data and support the conclusion that salicylate influences the electrical but not the mechanical properties of DPPC membranes. PMID:16216066

Phospholipid bilayer-perturbing properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes.

PubMed

Nogueira, Daniele Rubert; Mitjans, Montserrat; Busquets, M Antonia; Pérez, Lourdes; Vinardell, M Pilar

2012-08-14

Amino acid-based surfactants constitute an important class of natural surface-active biomolecules with an unpredictable number of industrial applications. To gain a better mechanistic understanding of surfactant-induced membrane destabilization, we assessed the phospholipid bilayer-perturbing properties of new cationic lysine-based surfactants. We used erythrocytes as biomembrane models to study the hemolytic activity of surfactants and their effects on cells' osmotic resistance and morphology, as well as on membrane fluidity and membrane protein profile with varying pH. The antihemolytic capacity of amphiphiles correlated negatively with the length of the alkyl chain. Anisotropy measurements showed that the pH-sensitive surfactants, with the positive charge on the α-amino group of lysine, significantly increased membrane fluidity at acidic conditions. SDS-PAGE analysis revealed that surfactants induced significant degradation of membrane proteins in hypo-osmotic medium and at pH 5.4. By scanning electron microscopy examinations, we corroborated the interaction of surfactants with lipid bilayer. We found that varying the surfactant chemical structure is a way to modulate the positioning of the molecule inside bilayer and, thus, the overall effect on the membrane. Our work showed that pH-sensitive lysine-based surfactants significantly disturb the lipid bilayer of biomembranes especially at acidic conditions, which suggests that these compounds are promising as a new class of multifunctional bioactive excipients for active intracellular drug delivery.

Aggregation of Aß(25-35) on DOPC and DOPC/DHA bilayers: an atomic force microscopy study.

PubMed

Sublimi Saponetti, Matilde; Grimaldi, Manuela; Scrima, Mario; Albonetti, Cristiano; Nori, Stefania Lucia; Cucolo, Annamaria; Bobba, Fabrizio; D'Ursi, Anna Maria

2014-01-01

β amyloid peptide plays an important role in both the manifestation and progression of Alzheimer disease. It has a tendency to aggregate, forming low-molecular weight soluble oligomers, higher-molecular weight protofibrillar oligomers and insoluble fibrils. The relative importance of these single oligomeric-polymeric species, in relation to the morbidity of the disease, is currently being debated. Here we present an Atomic Force Microscopy (AFM) study of Aβ(25-35) aggregation on hydrophobic dioleoylphosphatidylcholine (DOPC) and DOPC/docosahexaenoic 22∶6 acid (DHA) lipid bilayers. Aβ(25-35) is the smallest fragment retaining the biological activity of the full-length peptide, whereas DOPC and DOPC/DHA lipid bilayers were selected as models of cell-membrane environments characterized by different fluidity. Our results provide evidence that in hydrophobic DOPC and DOPC/DHA lipid bilayers, Aβ(25-35) forms layered aggregates composed of mainly annular structures. The mutual interaction between annular structures and lipid surfaces end-results into a membrane solubilization. The presence of DHA as a membrane-fluidizing agent is essential to protect the membrane from damage caused by interactions with peptide aggregates; to reduces the bilayer defects where the delipidation process starts.

Viscoelastic deformation of lipid bilayer vesicles.

PubMed

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

2015-10-07

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.

Viscoelastic deformation of lipid bilayer vesicles†

PubMed Central

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

2015-01-01

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. PMID:26268612

Cell membrane causes the lipid bilayers to behave as variable capacitors: A resonance with self-induction of helical proteins.

PubMed

Monajjemi, Majid

2015-12-01

Cell membrane has a unique feature of storing biological energies in a physiologically relevant environment. This study illustrates a capacitor model of biological cell membrane including DPPC structures. The electron density profile models, electron localization function (ELF) and local information entropy have been applied to study the interaction of proteins with lipid bilayers in the cell membrane. The quantum and coulomb blockade effects of different thicknesses in the membrane have also been specifically investigated. It has been exhibited the quantum effects can appear in a small region of the free space within the membrane thickness due to the number and type of phospholipid layers. In addition, from the viewpoint of quantum effects by Heisenberg rule, it is shown the quantum tunneling is allowed in some micro positions while it is forbidden in other forms of membrane capacitor systems. Due to the dynamical behavior of the cell membrane, its capacitance is not fixed which results a variable capacitor. In presence of the external fields through protein trance membrane or ions, charges exert forces that can influence the state of the cell membrane. This causes to appear the charge capacitive susceptibility that can resonate with self-induction of helical coils; the resonance of which is the main reason for various biological pulses. Copyright © 2015 Elsevier B.V. All rights reserved.

Interaction of Dendritic Polymers with Synthetic Lipid and Cell Membranes

NASA Astrophysics Data System (ADS)

Mecke, Almut; Hong, Seungpyo; Bielinska, Anna U.; Banaszak Holl, Mark M.; Orr, Bradford G.; Baker, James R., Jr.

2004-03-01

Polyamidoamine (PAMAM) dendrimers are promising candidates for the development of nanoscale therapeutic transport agents. Here we present studies on dendrimer-membrane interactions leading to a better understanding of possible uptake mechanisms into cells. Using synthetic lipid and natural cell membranes as model systems it is shown that the effect of PAMAM dendrimers on a membrane strongly depends on the dendrimer generation, architecture and chemical properties of the branch end groups. Atomic force microscopy data indicates that generation 7 dendrimers have the ability to form small ( 10-100 nm) holes in a lipid bilayer. When dendrimers with otherwise identical chemical properties are arranged in a covalently linked cluster, no hole formation occurs. Dendrimer-lipid micelle formation is proposed and investigated as a possible mechanism for this behavior. Smaller dendrimers (generation 5) have a greatly reduced ability to remove lipid molecules from a bilayer. In addition to the size of the dendrimer, the charge of the branch end groups plays a significant role for dendrimer-membrane interactions. These results agree well with biological studies using cultured cells and point to a new mechanism of specific targeting and uptake into cells.

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

PubMed

Chadda, R; Robertson, J L

2016-01-01

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F - ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl - /H + antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known. © 2016 Elsevier Inc. All rights reserved.

Studies of molecular diffusion in single-supported bilayer lipid membranes at high hydration by quasielastic neutron scattering

NASA Astrophysics Data System (ADS)

Bai, M.; Miskowiec, A.; Wang, S.-K.; Taub, H.; Hansen, F. Y.; Jenkins, T.; Tyagi, M.; Neumann, D. A.; Diallo, S. O.; Mamontov, E.; Herwig, K. W.

2011-03-01

Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently obtained quasielastic neutron spectra from single-supported bilayer lipid membranes using the backscattering spectrometer BASIS at the Spallation Neutron Source. Protonated DMPC membranes were deposited onto Si O2 -coated Si(100) substrates and characterized by AFM. Analysis of their neutron spectra shows evidence of a relatively broad Lorentzian component that we associate with bulk-like water above a freezing temperature of ~ 267 K. At lower temperatures, the spectra differ qualitatively from that of bulk supercooled water, a behavior that we attribute to water bound to the membrane. We also find evidence of a narrow Lorentzian component that we tentatively identify with a slower motion (time scale ~ 1 ns) associated with conformational changes of the alkyl tails of the lipid molecules. Supported by NSF Grant No. DMR-0705974.

Aspirin Increases the Solubility of Cholesterol in Lipid Membranes

NASA Astrophysics Data System (ADS)

Alsop, Richard; Barrett, Matthew; Zheng, Sonbo; Dies, Hannah; Rheinstadter, Maikel

2014-03-01

Aspirin (ASA) is often prescribed for patients with high levels of cholesterol for the secondary prevention of myocardial events, a regimen known as the Low-Dose Aspirin Therapy. We have recently shown that Aspirin partitions in lipid bilayers. However, a direct interplay between ASA and cholesterol has not been investigated. Cholesterol is known to insert itself into the membrane in a dispersed state at moderate concentrations (under ~37.5%) and decrease fluidity of membranes. We prepared model lipid membranes containing varying amounts of both ASA and cholesterol molecules. The structure of the bilayers as a function of ASA and cholesterol concentration was determined using high-resolution X-ray diffraction. At cholesterol levels of more than 40mol%, immiscible cholesterol plaques formed. Adding ASA to the membranes was found to dissolve the cholesterol plaques, leading to a fluid lipid bilayer structure. We present first direct evidence for an interaction between ASA and cholesterol on the level of the cell membrane.

Revealing the membrane-bound structure of neurokinin A using neutron diffraction

NASA Astrophysics Data System (ADS)

Darkes, Malcolm J. M.; Hauss, Thomas; Dante, Silvia; Bradshaw, Jeremy P.

2000-03-01

Neurokinin A (or substance K) belongs to the tachykinin family, a group of small amphipathic peptides that bind to specific membrane-embedded, G-protein coupled receptors. The agonist/receptor complex is quaternary in nature because the receptor binding sites are thought to be located within the lipid bilayer and because the role of water cannot be ignored. The cell membrane acts as a solvent to accumulate peptide and an inducer of peptide secondary structure. The three-dimensional shape that the peptide assumes when associated to the cell membrane will be an important parameter with regards to the receptor selectivity and affinity. Neutron diffraction measurements were carried out in order to define the location of the N-terminus of the peptide in synthetic phospholipid multi-bilayer stacks.

Study of supported bilayer lipid membranes for use in chemo-electric energy conversion via active proton transport

NASA Astrophysics Data System (ADS)

Sarles, Stephen A.; Sundaresan, Vishnu B.; Leo, Donald J.

2007-09-01

Bilayer lipid membranes (BLMs) have been studied extensively due to functional and structural similarities to cell membranes, fostering research to understand ion-channel protein functions, measure bilayer mechanical properties, and identify self-assembly mechanisms. BLMs have traditionally been formed across single pores in substrates such as PTFE (Teflon). The incorporation of ion-channel proteins into the lipid bilayer enables the selective transfer of ions and fluid through the BLM. Processes of this nature have led to the measurement of ion current flowing across the lipid membrane and have been used to develop sensors that signal the presence of a particular reactant (glucose, urea, penicillin), improve drug recognition in cells, and develop materials capable of creating chemical energy from light. Recent research at Virginia Tech has shown that the incorporation of proton transporters in a supported BLM formed across an array of pores can convert chemical energy available in the adenosine triphosphate (ATP) into electricity. Experimental results from this work show that the system-named Biocell-is capable of developing 2µW/cm2 of membrane area with 15μl of ATPase. Efforts to increase the power output and conversion efficiency of this process while moving toward a packaged device present a unique engineering problem. The bilayer, as host to the active proton transporters, must therefore be formed evenly across a porous substrate, remain stable and yet fluid-like for protein interaction, and exhibit a large seal resistance. This article presents the ongoing work to characterize the Biocell using impedance analysis. Electrical impedance spectroscopy (EIS) is used to study the effect of adding ATPase proteins to POPS:POPE bilayer lipid membranes and correlate structural changes evident in the impedance data to the energy-conversion capability of various partial and whole Biocell assemblies. The specific membrane resistance of a pure BLM drops from 40-120kΩ•cm2 to only a few hundred Ω•cm2 upon reconstitution of ATPase proteins. Power characterization indicates that ATP hydrolysis may result in charging of the silver-silver chloride electrodes.

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

2012-03-07

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 due to the interaction of CPZ with the membranes. © 2012 American Chemical Society

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.

DSC and EPR investigations on effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within lipid bilayer membrane.

PubMed

Zhao, Lingyun; Feng, Si-Shen; Kocherginsky, Nikolai; Kostetski, Iouri

2007-06-29

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) were applied to investigate effects of cholesterol component on molecular interactions between paclitaxel, which is one of the best antineoplastic agents found from nature, and dipalmitoylphosphatidylcholine (DPPC) within lipid bilayer vesicles (liposomes), which could also be used as a model cell membrane. DSC analysis showed that incorporation of paclitaxel into the DPPC bilayer causes a reduction in the cooperativity of bilayer phase transition, leading to a looser and more flexible bilayer structure. Including cholesterol component in the DPPC/paclitaxel mixed bilayer can facilitate the molecular interaction between paclitaxel and lipid and make the tertiary system more stable. EPR analysis demonstrated that both of paclitaxel and cholesterol have fluidization effect on the DPPC bilayer membranes although cholesterol has more significant effect than paclitaxel does. The reduction kinetics of nitroxides by ascorbic acid showed that paclitaxel can inhibit the reaction by blocking the diffusion of either the ascorbic acid or nitroxide molecules since the reaction is tested to be a first order one. Cholesterol can remarkably increase the reduction reaction speed. This research may provide useful information for optimizing liposomal formulation of the drug as well as for understanding the pharmacology of paclitaxel.

Nonenzymatic Reactions above Phospholipid Surfaces of Biological Membranes: Reactivity of Phospholipids and Their Oxidation Derivatives

PubMed Central

Solís-Calero, Christian; Ortega-Castro, Joaquín; Frau, Juan; Muñoz, Francisco

2015-01-01

Phospholipids play multiple and essential roles in cells, as components of biological membranes. Although phospholipid bilayers provide the supporting matrix and surface for many enzymatic reactions, their inherent reactivity and possible catalytic role have not been highlighted. As other biomolecules, phospholipids are frequent targets of nonenzymatic modifications by reactive substances including oxidants and glycating agents which conduct to the formation of advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs). There are some theoretical studies about the mechanisms of reactions related to these processes on phosphatidylethanolamine surfaces, which hypothesize that cell membrane phospholipids surface environment could enhance some reactions through a catalyst effect. On the other hand, the phospholipid bilayers are susceptible to oxidative damage by oxidant agents as reactive oxygen species (ROS). Molecular dynamics simulations performed on phospholipid bilayers models, which include modified phospholipids by these reactions and subsequent reactions that conduct to formation of ALEs and AGEs, have revealed changes in the molecular interactions and biophysical properties of these bilayers as consequence of these reactions. Then, more studies are desirable which could correlate the biophysics of modified phospholipids with metabolism in processes such as aging and diseases such as diabetes, atherosclerosis, and Alzheimer's disease. PMID:25977746

Development of an implantable synthetic membrane for the treatment of preterm premature rupture of fetal membranes.

PubMed

Roman, Sabiniano; Bullock, Anthony J; Anumba, Dilly O; MacNeil, Sheila

2016-02-01

Preterm premature rupture of fetal membranes is a very common condition leading to premature labour of a non viable fetus. Significant morbidities may occur when preterm premature rupture of fetal membranes management is attempted to prolong the pregnancy for fetal maturation. Reducing the rate of loss of amniotic fluid and providing a barrier to bacterial entry may allow the pregnancy to continue to term, avoiding complications. Our aim is to develop a synthetic biocompatible membrane to form a distensible barrier for cervical closure which acts to reduce fluid loss and provide a surface for epithelial ingrowth to help repair the damaged membranes. Therefore, a bilayer membrane was developed using an electrospinning technique of combining two FDA-approved polymers, poly-L-lactic acid (PLA) and polyurethane (Z3) polymer. This was compared to a plain electrospun Z3 membrane. The physical and mechanical properties were assessed using scanning electron microscope images and a BOSE tensiometer, respectively, and compared to native fetal membranes. The performance of the membranes in preventing fluid loss was assessed by measuring their ability to support a column of water. Finally the ability of the membranes to support cell ingrowth was assessed by culturing adipose-derived stem cells on the membranes for two weeks and assessing metabolic activity after 7 and 14 days. The physical properties of the bilayer were similar to that of the native fetal membranes and it was resistant to fluid penetration. This bilayer membrane presented mechanical properties close to those for fetal membranes and showed elastic distention, which may be crucial for progress of the pregnancy. The membrane was also able to retain surgical sutures. In addition, it also supported the attachment and growth of adipose-derived stem cells for two weeks. In conclusion, this membrane may prove a useful approach in the treatment of preterm premature rupture of fetal membranes and now merits further investigation. © The Author(s) 2015.

Molecular machines open cell membranes

NASA Astrophysics Data System (ADS)

García-López, Víctor; Chen, Fang; Nilewski, Lizanne G.; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B.; Robinson, Jacob T.; Wang, Gufeng; Pal, Robert; Tour, James M.

2017-08-01

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

Molecular machines open cell membranes.

PubMed

García-López, Víctor; Chen, Fang; Nilewski, Lizanne G; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B; Robinson, Jacob T; Wang, Gufeng; Pal, Robert; Tour, James M

2017-08-30

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

GPI-anchored protein organization and dynamics at the cell surface

PubMed Central

Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-01-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. PMID:26394904

GPI-anchored protein organization and dynamics at the cell surface.

PubMed

Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-02-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

The Physical Properties of Ceramides in Membranes.

PubMed

Alonso, Alicia; Goñi, Félix M

2018-05-20

Ceramides are sphingolipids containing a sphingosine or a related base, to which a fatty acid is linked through an amide bond. When incorporated into a lipid bilayer, ceramides exhibit a number of properties not shared by almost any other membrane lipid: Ceramides ( a) are extremely hydrophobic and thus cannot exist in suspension in aqueous media; ( b) increase the molecular order (rigidity) of phospholipids in membranes; ( c) give rise to lateral phase separation and domain formation in phospholipid bilayers; ( d) possess a marked intrinsic negative curvature that facilitates formation of inverted hexagonal phases; ( e) make bilayers and cell membranes permeable to small and large (i.e., protein-size) solutes; and ( f) promote transmembrane (flip-flop) lipid motion. Unfortunately, there is hardly any link between the physical studies reviewed here and the mass of biological and clinical studies on the effects of ceramides in health and disease.

Compression-triggered instabilities of multi-layer systems: From thin elastic membranes to lipid bilayers on flexible substrates

NASA Astrophysics Data System (ADS)

Stone, Howard A.

2013-03-01

Instabilities are triggered when elastic materials are subjected to compression. We explore new features of two distinct systems of this type. First, we describe a two-layer polymeric system under biaxial compressive stress, which exhibits a repetitive wrinkle-to-fold transition that subsequently generates a hierarchical network of folds during reorganization of the stress field. The folds delineate individual domains, and each domain subdivides into smaller ones over multiple generations. By modifying the boundary conditions and geometry, we demonstrate control over the final network morphology. Some analogies to the venation pattern of leaves are indicated. Second, motivated by the confined configurations common to cells, which are wrapped in lipid bilayer membranes, we study a lipid bilayer, coupled to an elastic sheet, and demonstrate that, upon straining, the confined lipid membrane is able to passively regulate its area. In particular, by stretching the elastic support, the bilayer laterally expands without rupture by fusing adhered lipid vesicles; upon compression, lipid tubes grow out of the membrane plane, thus reducing its area. These transformations are reversible, as we show using cycles of expansion and compression, and closely reproduce membrane processes found in cells during area regulation. The two distinct systems illustrate the influence of the substrate on finite amplitude shape changes, for which we describe the time-dependent shape evolution as the stress relaxes. This talk describes joint research with Manouk Abkarian, Marino Arroyo, Pilnam Kim, Mohammad Rahimi and Margarita Staykova.

In-Plane Correlations in a Polymer-Supported Lipid Membrane Measured by Off-Specular Neutron Scattering

NASA Astrophysics Data System (ADS)

Jablin, Michael S.; Zhernenkov, Mikhail; Toperverg, Boris P.; Dubey, Manish; Smith, Hillary L.; Vidyasagar, Ajay; Toomey, Ryan; Hurd, Alan J.; Majewski, Jaroslaw

2011-04-01

Polymer-supported single lipid bilayers are models to study configurations of cell membranes. We used off-specular neutron scattering to quantify in-plane height-height correlations of interfacial fluctuations of such a lipid bilayer. As temperature decreased from 37°C to 25°C, the polymer swells and the polymer-supported lipid membrane deviates from its initially nearly planar structure. A correlation length characteristic of capillary waves changes from 30μm at 37°C to 11μm at 25°C, while the membrane bending rigidity remains roughly constant in this temperature range.

Does transbilayer diffusion have a role in membrane transport of drugs?

PubMed Central

Balaz, Stefan

2012-01-01

The existing consensus on coexistence of transbilayer diffusion and carrier-mediated transport as two main mechanisms for drugs crossing biological membranes was recently challenged by a systems biology group. Their transporters-only hypothesis is examined in this article using published experimental evidence. The main focus is on the key claim of their hypothesis, stating that ‘the drug molecules cross pure phospholipid bilayers through transient pores that cannot form in the bilayers of cell membranes, and thus transbilayer drug transport does not exist in cells’. The analysis shows that the prior consensus remains a valid scientific view of the membrane transport of drugs. PMID:22705388

Spontaneous vesicle formation at lipid bilayer membranes.

PubMed

Edwards, D A; Schneck, F; Zhang, I; Davis, A M; Chen, H; Langer, R

1996-09-01

Unilamellar vesicles are observed to form spontaneously at planar lipid bilayers agitated by exothermic chemical reactions. The membrane-binding reaction between biotin and streptavidin, two strong transmembrane neutralization reactions, and a weak neutralization reaction involving an "antacid" buffer, all lead to spontaneous vesicle formation. This formation is most dramatic when a viscosity differential exists between the two phases bounding the membrane, in which case vesicles appear exclusively in the more viscous phase. A hydrodynamic analysis explains the phenomenon in terms of a membrane flow driven by liberated reaction energy, leading to vesicle formation. These results suggest that energy liberated by intra- and extracellular chemical reactions near or at cell and internal organelle membranes can play an important role in vesicle formation, membrane agitation, or enhanced transmembrane mass transfer.

Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suwalsky, Mario, E-mail: msuwalsk@udec.cl; Zambrano, Pablo; Mennickent, Sigrid

Research highlights: {yields} PPA is a common ingredient in cough-cold medication and appetite suppressants. {yields} Reports on its effects on human erythrocytes are very scarce. {yields} We found that PPA induced in vitro morphological changes to human erythrocytes. {yields} PPA interacted with isolated unsealed human erythrocyte membranes. {yields} PPA interacted with class of lipid present in the erythrocyte membrane outer monolayer. -- Abstract: Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada.more » Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 {mu}M; (c) X-ray diffraction studies showed that PPA in the 0.1-0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.« less

Electrodeformation of multi-bilayer spherical concentric membranes by AC electric fields

NASA Astrophysics Data System (ADS)

Lira-Escobedo, J.; Arauz-Lara, J.; Aranda-Espinoza, H.; Adlerz, K.; Viveros-Mendez, P. X.; Aranda-Espinoza, S.

2017-09-01

It is now well established that external stresses alter the behaviour of cells, where such alterations can be as profound as changes in gene expression. A type of stresses of particular interest are those due to alternating-current (AC) electric fields. The effect of AC fields on cells is still not well understood, in particular it is not clear how these fields affect the cell nucleus and other organelles. Here, we propose that one possible mechanism is through the deformation of the membranes. In order to investigate the effect of AC fields on the morphological changes of the cell organelles, we modelled the cell as two concentric bilayer membranes. This model allows us to obtain the deformations induced by the AC field by balancing the elastic energy and the work done by the Maxwell stresses. Morphological phase diagrams are obtained as a function of the frequency and the electrical properties of the media and membranes. We demonstrate that the organelle shapes can be changed without modifying the shape of the external cell membrane and that the organelle deformation transitions can be used to measure, for example, the conductivity of the nucleus.

Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells

PubMed Central

Karatekin, Erdem; Rothman, James E.

2013-01-01

Many biological processes rely on membrane fusion, therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorsecence microscopy. Unlike most previous attempts, fusion here is dependent on SNAP25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of (i) bilayers covered with a thin layer of poly(ethylene glycol) to control bilayer-bilayer and bilayer-substrate interactions, (ii) microfluidic flow channels which presents many advantages such as the removal of non-specifically bound liposomes by flow. The protocol takes 6–8 days to complete. Analysis can take up to two weeks. PMID:22517259

Effect of acetone accumulation on structure and dynamics of lipid membranes studied by molecular dynamics simulations.

PubMed

Posokhov, Yevgen O; Kyrychenko, Alexander

2013-10-01

The modulation of the properties and function of cell membranes by small volatile substances is important for many biomedical applications. Despite available experimental results, molecular mechanisms of action of inhalants and organic solvents, such as acetone, on lipid membranes remain not well understood. To gain a better understanding of how acetone interacts with membranes, we have performed a series of molecular dynamics (MD) simulations of a POPC bilayer in aqueous solution in the presence of acetone, whose concentration was varied from 2.8 to 11.2 mol%. The MD simulations of passive distribution of acetone between a bulk water phase and a lipid bilayer show that acetone favors partitioning into the water-free region of the bilayer, located near the carbonyl groups of the phospholipids and at the beginning of the hydrocarbon core of the lipid membrane. Using MD umbrella sampling, we found that the permeability barrier of ~0.5 kcal/mol exists for acetone partitioning into the membrane. In addition, a Gibbs free energy profile of the acetone penetration across a bilayer demonstrates a favorable potential energy well of -3.6 kcal/mol, located at 15-16Å from the bilayer center. The analysis of the structural and dynamics properties of the model membrane revealed that the POPC bilayer can tolerate the presence of acetone in the concentration range of 2.8-5.6 mol%. The accumulation of the higher acetone concentration of 11.2 mol% results, however, in drastic disordering of phospholipid packing and the increase in the membrane fluidity. The acetone molecules push the lipid heads apart and, hence, act as spacers in the headgroup region. This effect leads to the increase in the average headgroup area per molecule. In addition, the acyl tail region of the membrane also becomes less dense. We suggest, therefore, that the molecular mechanism of acetone action on the phospholipid bilayer has many common features with the effects of short chain alcohols, DMSO, and chloroform. Copyright © 2013 Elsevier Ltd. All rights reserved.

Organization of lipids in fiber-cell plasma membranes of the eye lens.

PubMed

Subczynski, Witold K; Mainali, Laxman; Raguz, Marija; O'Brien, William J

2017-03-01

The plasma membrane together with the cytoskeleton forms the only supramolecular structure of the matured fiber cell which accounts for mostly all fiber cell lipids. The purpose of this review is to inform researchers about the importance of the lipid bilayer portion of the lens fiber cell plasma membranes in the maintaining lens homeostasis, and thus protecting against cataract development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae

PubMed Central

Henderson, Clark M.

2014-01-01

Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated. PMID:24610851

Observing a model ion channel gating action in model cell membranes in real time in situ: membrane potential change induced alamethicin orientation change.

PubMed

Ye, Shuji; Li, Hongchun; Wei, Feng; Jasensky, Joshua; Boughton, Andrew P; Yang, Pei; Chen, Zhan

2012-04-11

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split α/3(10)-helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer and was measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH = 6.7 the α-helix at the N-terminus and the 3(10)-helix at the C-terminus tilt at about 72° (θ(1)) and 50° (θ(2)) versus the surface normal, respectively. When pH increases to 11.9, θ(1) and θ(2) decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ(1) for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and the bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes. © 2012 American Chemical Society

Indentation of Graphene-Covered Atomic Force Microscopy Probe Across a Lipid Bilayer Membrane: Effect of Tip Shape, Size, and Surface Hydrophobicity.

PubMed

Lv, Kang; Li, Yinfeng

2018-06-21

Understanding the interaction of graphene with cell membranes is crucial to the development of graphene-based biological applications and the management of graphene safety issues. To help reveal the key factors controlling the interaction between graphene and cell membranes, here we adopt the dissipative particle dynamics method to analyze the evolution of interaction force and free energy as the graphene-covered atomic force microscopy (AFM) probe indents across a lipid bilayer. The simulation results show that the graphene-covered AFM probe can cause severe deformation of the cell membrane which drives the lipid molecule to adsorb and diffuse at the surface of graphene. The breakthrough force and free energy are calculated to study the effects of the tip shape, size, and surface hydrophobicity on the piercing behaviors of graphene-covered AFM. In addition, the deformation of cell membrane can decrease the dependency of the breakthrough force on the tip shape. The analysis of surface functionalization suggests that the horizontal patterns on graphene can change the preferred orientation in the penetration process, but the vertical patterns on graphene may disrupt the cell membrane. What's more, the bending stiffness of graphene has little influence on the penetration process as graphene pierces into the cell membrane. These results provide useful guidelines for the molecular design of graphene materials with controllable cell penetrability.

Charging the quantum capacitance of graphene with a single biological ion channel.

PubMed

Wang, Yung Yu; Pham, Ted D; Zand, Katayoun; Li, Jinfeng; Burke, Peter J

2014-05-27

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents.

Charging the Quantum Capacitance of Graphene with a Single Biological Ion Channel

PubMed Central

2015-01-01

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents. PMID:24754625

Properties of Fiber Cell Plasma Membranes Isolated from the Cortex and Nucleus of the Porcine Eye Lens

PubMed Central

Mainali, Laxman; Raguz, Marija; O’Brien, William J.; Subczynski, Witold K.

2012-01-01

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region. PMID:22326289

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

Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

PubMed

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

2014-10-01

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.

Effect of Amphotericin B antibiotic on the properties of model lipid membrane

NASA Astrophysics Data System (ADS)

Kiryakova, S.; Dencheva-Zarkova, M.; Genova, J.

2014-12-01

Model membranes formed from natural and synthetic lipids are an interesting object for scientific investigations due to their similarity to biological cell membrane and their simple structure with controlled composition and properties. Amphotericin B is an important polyene antifungal antibiotic, used for treatment of systemic fungal infections. It is known from the literature that the studied antibiotic has a substantial effect on the transmembrane ionic channel structures. When applied to the lipid membranes it has the tendency to create pores and in this way to affect the structure and the properties of the membrane lipid bilayer. In this work the thermally induced shape fluctuations of giant quasi-spherical liposomes have been used to study the influence of polyene antibiotic amphotericin B on the elastic properties of model lipid membranes. It have been shown experimentally that the presence of 3 mol % of AmB in the lipid membrane reduces the bending elasticity of the lipid membrane for both studied cases: pure SOPC membrane and mixed SOPC-Cholesterol membrane. Interaction of the amphotericin B with bilayer lipid membranes containing channels have been studied in this work. Model membranes were self-assembled using the patch-clamp and tip-dip patch clamp technique. We have found that amphotericin B is an ionophore and reduces the resistance of the lipid bilayer.

Effect of Membrane Tension on the Electric Field and Dipole Potential of Lipid Bilayer Membrane

PubMed Central

Warshaviak, Dora Toledo; Muellner, Michael J.; Chachisvilis, Mirianas

2011-01-01

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45 mV in the physiologically relevant range of membrane tension values (0 to 15 dyn/cm). The electrostatic field exhibits a peak (~0.8×109 V/m) near the water/lipid interface which shifts by 0.9 Å towards the bilayer center at 15 dyn/cm. Maximum membrane tension of 15 dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states. PMID:21722624

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 membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate. PMID:23746513

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

PubMed

Mossberg, Ann-Kristin; Puchades, Maja; Halskau, Øyvind; Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

2010-02-23

Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

The Role of Nanoparticle Surface Functionality in the Disruption of Model Cell Membranes

PubMed Central

Moghadam, Babak Y.; Hou, Wen-Che; Corredor, Charlie; Westerhoff, Paul; Posner, Jonathan D.

2012-01-01

Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of engineered nanomaterials (ENMs) with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assess the effect of varying the surface functionality and concentration of 10 nm-diameter gold (Au) and titanium dioxide (TiO2) ENMs on the disruption of negatively charged lipid bilayer vesicles (liposomes) using a dye leakage assay. Our findings show that Au ENMs having both positive and negative surface charge induce leakage that reaches a steady state after several hours. Positively charged particles with identical surface functionality and different core composition show similar leakage effects and result in faster and greater leakage than negatively charged particles, which suggests that surface functionality, not particle core composition, is a critical factor in determining the interaction between ENMs and lipid bilayers. The results suggest that particles permanently adsorb to bilayers and that only one positively charged particle is required to disrupt a liposome and trigger leakage of its entire contents in contrast to mellitin molecules, the most widely studied membrane lytic peptide, which requires hundred of molecules to generate leakage. PMID:22921268

Brownian dynamics simulations of lipid bilayer membrane with hydrodynamic interactions in LAMMPS

NASA Astrophysics Data System (ADS)

Fu, Szu-Pei; Young, Yuan-Nan; Peng, Zhangli; Yuan, Hongyan

2016-11-01

Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiencies have been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4 6 nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane (such as gel-fluid-gas phase transitions of lipids, diffusion, and bending rigidity). In this work we implement such interaction potential in LAMMPS to simulate large-scale lipid systems such as vesicles and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for red blood cell (RBC) dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. We focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with varying enclosed volume; 2. RBC shape transitions with different enclosed volume. This work is funded by NSF under Grant DMS-1222550.

Brownian dynamics simulations of lipid bilayer membrane with hydrodynamic interactions in LAMMPS

NASA Astrophysics Data System (ADS)

Fu, Szu-Pei; Young, Yuan-Nan; Peng, Zhangli; Yuan, Hongyan

Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiency has been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4 6 nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane (such as gel-fluid-gas phase transitions of lipids, diffusion, and bending rigidity). In this work we implement such interaction potential in LAMMPS to simulate large-scale lipid systems such as vesicles and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for red blood cell (RBC) dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. We focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with varying enclosed volume; 2. RBC shape transitions with different enclosed volume.

Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

PubMed

Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

2015-04-01

Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

Transmembrane peptides as sensors of the membrane physical state

NASA Astrophysics Data System (ADS)

Piotto, Stefano; Di Biasi, Luigi; Sessa, Lucia; Concilio, Simona

2018-05-01

Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state suggested a new approach for the discovery of antimicrobial peptides.

Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes.

PubMed

Lewis, Ruthven N A H; Kiricsi, Monika; Prenner, Elmar J; Hodges, Robert S; McElhaney, Ronald N

2003-01-21

Cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)), a synthetic tetradecameric ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S (GS), retains the strong antimicrobial activity of GS but is 15-20 times less hemolytic. To characterize its interaction with lipid membranes and to understand the molecular basis of its capacity to lyse bacterial cells, in preference to erythrocytes, we have investigated the interactions of GS14dK(4) with detergent micelles and with lipid bilayer model membranes by Fourier transform infrared spectroscopy and compared our results with those of a similar study of GS [Lewis, R. N. A. H., et al. (1999) Biochemistry 38, 15193-15203]. In both aqueous and organic solvent solutions, GS14dK(4) adopts a beta-sheet conformation that is somewhat distorted and more sensitive to the polarity of its environment than GS. Like GS, GS14dK(4) is completely or partially excluded from gel-state lipid bilayers but interacts strongly with liquid-crystalline lipid bilayers and detergent micelle, and interacts more strongly with more fluid liquid-crystalline lipid systems. However, its interactions are more strongly influenced by membrane lipid order and fluidity, and unlike GS, it is essentially excluded from cholesterol-containing phospholipid bilayers. Also, GS14dK(4) is excluded from cationic lipid bilayers, but partitions more strongly and/or penetrates more deeply into anionic lipid bilayers than into those composed of either zwitterionic or nonionic lipids. Anionic lipids also facilitate GS14dK(4) interactions with multicomponent lipid bilayers which are predominantly zwitterionic or nonionic. Although GS14dK(4) generally penetrates and/or partitions into zwitterionic or uncharged lipid bilayers less strongly than does GS, its greater size and altered distribution of positive charges make it intrinsically more perturbing with regard to membrane organization once associated with lipid bilayers. This fact, combined with its relatively strong interactions with anionic phospholipids, may explain why GS14dK(4) retains relatively high antimicrobial activity. However, its low hemolytic activity is probably largely attributable to its low propensity to penetrate and/or partition into cholesterol-containing zwitterionic lipid membranes.

Nanoscale investigation of the interaction of colistin with model phospholipid membranes by Langmuir technique, and combined infrared and force spectroscopies.

PubMed

Freudenthal, Oona; Quilès, Fabienne; Francius, Grégory; Wojszko, Kamila; Gorczyca, Marcelina; Korchowiec, Beata; Rogalska, Ewa

2016-11-01

Colistin (Polymyxin E), an antimicrobial peptide, is increasingly put forward as salvage for severe multidrug-resistant infections. Unfortunately, colistin is potentially toxic to mammalian cells. A better understanding of the interaction with specific components of the cell membranes may be helpful in controlling the factors that may enhance toxicity. Here, we report a physico-chemical study of model phospholipid (PL) mono- and bilayers exposed to colistin at different concentrations by Langmuir technique, atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The effect of colistin on chosen PL monolayers was examined. Insights into the topographical and elastic changes in the PL bilayers within time after peptide injection are presented via AFM imaging and force spectra. Finally, changes in the PL bilayers' ATR-FTIR spectra as a function of time within three bilayer compositions, and the influence of colistin on their spectral fingerprint are examined together with the time-evolution of the Amide II and νCO band integrated intensity ratios. Our study reveals a great importance in the role of the PL composition as well as the peptide concentration on the action of colistin on PL model membranes. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

PubMed

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

Engineering antimicrobial peptides with improved antimicrobial and hemolytic activities.

PubMed

Zhao, Jun; Zhao, Chao; Liang, Guizhao; Zhang, Mingzhen; Zheng, Jie

2013-12-23

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.

Hydrodynamic collective effects of active protein machines in solution and lipid bilayers

PubMed Central

Mikhailov, Alexander S.; Kapral, Raymond

2015-01-01

The cytoplasm and biomembranes in biological cells contain large numbers of proteins that cyclically change their shapes. They are molecular machines that can function as molecular motors or carry out various other tasks in the cell. Many enzymes also undergo conformational changes within their turnover cycles. We analyze the advection effects that nonthermal fluctuating hydrodynamic flows induced by active proteins have on other passive molecules in solution or membranes. We show that the diffusion constants of passive particles are enhanced substantially. Furthermore, when gradients of active proteins are present, a chemotaxis-like drift of passive particles takes place. In lipid bilayers, the effects are strongly nonlocal, so that active inclusions in the entire membrane contribute to local diffusion enhancement and the drift. All active proteins in a biological cell or in a membrane contribute to such effects and all passive particles, and the proteins themselves, will be subject to them. PMID:26124140

Do trehalose and dimethyl sulfoxide affect intermembrane forces?

PubMed

Pincet, F; Perez, E; Wolfe, J

1994-12-01

The sugar trehalose is produced in some organisms that survive dehydration and desiccation, and it preserves the integrity of membranes in model systems exposed to dehydration and freezing. Dimethyl sulfoxide, a solute which permeates membranes, is added to cell suspensions in many protocols for cryopreservation. Using a surface forces apparatus, we measured the very large, short-range repulsion between phosphatidylcholine bilayers in water and in solutions of trehalose, sorbitol, and dimethyl-sulfoxide. To the resolution of the technique, the force-distance curves between bilayers are unchanged by the addition of trehalose or sorbitol in concentrations exceeding 1 kmol.m-3. A relatively small increase in adhesion in the presence of trehalose and sorbitol solutions may be explained by their osmotic effects. The partitioning of trehalose between aqueous solutions and lamellar phases of dioleylphosphatidylcholine was measured gravimetrically. The amount of trehalose that preferentially adsorbs near membrane surfaces is at most small. The presence of dimethyl sulfoxide in water (1:2 by volume) makes very little difference to the short-range interaction between deposited bilayers, but it sometimes perturbs them in ways that vary among experiments: free bilayers and/or fusion of the deposited bilayers were each observed in about one-third of the experiments.

Synthesis and Characterization of Novel Anchorlipids for Tethered Bilayer Lipid Membranes.

PubMed

Andersson, Jakob; Knobloch, Jacqueline J; Perkins, Michael V; Holt, Stephen A; Köper, Ingo

2017-05-09

Tethered bilayer lipid membranes are versatile solid-supported model membrane systems. Core to these systems is an anchorlipid that covalently links a lipid bilayer to a support. The molecular structure of these lipids can have a significant impact on the properties of the resulting bilayer. Here, the synthesis of anchorlipids containing ester groups in the tethering part is described. The lipids are used to form bilayer membranes, and the resulting structures are compared with membranes formed using conventional anchorlipids or sparsely tethered membranes. All membranes showed good electrical sealing properties; the disulphide-terminated anchorlipids could be used in a sparsely tethered system without significantly reducing the sealing properties of the lipid bilayers. The sparsely tethered systems also allowed for higher ion transport across the membrane, which is in good correlation with higher hydration of the spacer region as seen by neutron scattering.

Inhibiting host-pathogen interactions using membrane-based nanostructures.

PubMed

Bricarello, Daniel A; Patel, Mira A; Parikh, Atul N

2012-06-01

Virulent strains of bacteria and viruses recognize host cells by their plasma membrane receptors and often exploit the native translocation machinery to invade the cell. A promising therapeutic concept for early interruption of pathogen infection is to subvert this pathogenic trickery using exogenously introduced decoys that present high-affinity mimics of cellular receptors. This review highlights emerging applications of molecularly engineered lipid-bilayer-based nanostructures, namely (i) functionalized liposomes, (ii) supported colloidal bilayers or protocells and (iii) reconstituted lipoproteins, which display functional cellular receptors in optimized conformational and aggregative states. These decoys outcompete host cell receptors by preferentially binding to and neutralizing virulence factors of both bacteria and viruses, thereby promising a new approach to antipathogenic therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structured Water Layers Adjacent to Biological Membranes

PubMed Central

Higgins, Michael J.; Polcik, Martin; Fukuma, Takeshi; Sader, John E.; Nakayama, Yoshikazu; Jarvis, Suzanne P.

2006-01-01

Water amid the restricted space of crowded biological macromolecules and at membrane interfaces is essential for cell function, though the structure and function of this “biological water” itself remains poorly defined. The force required to remove strongly bound water is referred to as the hydration force and due to its widespread importance, it has been studied in numerous systems. Here, by using a highly sensitive dynamic atomic force microscope technique in conjunction with a carbon nanotube probe, we reveal a hydration force with an oscillatory profile that reflects the removal of up to five structured water layers from between the probe and biological membrane surface. Further, we find that the hydration force can be modified by changing the membrane fluidity. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine gel (Lβ) phase bilayers, each oscillation in the force profile indicates the force required to displace a single layer of water molecules from between the probe and bilayer. In contrast, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine fluid (Lα) phase bilayers at 60°C and 1,2-dioleoyl-sn-glycero-3-phosphocholine fluid (Lα) phase bilayers at 24°C seriously disrupt the molecular ordering of the water and result predominantly in a monotonic force profile. PMID:16798815

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models.

PubMed

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2017-01-29

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40-50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein diffusion in plant cell plasma membranes: the cell-wall corral.

PubMed

Martinière, Alexandre; Runions, John

2013-01-01

Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.

PubMed

Mason, R Preston; Jacob, Robert F; Shrivastava, Sandeep; Sherratt, Samuel C R; Chattopadhyay, Amitabha

2016-12-01

Cholesterol crystalline domains characterize atherosclerotic membranes, altering vascular signaling and function. Omega-3 fatty acids reduce membrane lipid peroxidation and subsequent cholesterol domain formation. We evaluated non-peroxidation-mediated effects of eicosapentaenoic acid (EPA), other TG-lowering agents, docosahexaenoic acid (DHA), and other long-chain fatty acids on membrane fluidity, bilayer width, and cholesterol domain formation in model membranes. In membranes prepared at 1.5:1 cholesterol-to-phospholipid (C/P) mole ratio (creating pre-existing domains), EPA, glycyrrhizin, arachidonic acid, and alpha linolenic acid promoted the greatest reductions in cholesterol domains (by 65.5%, 54.9%, 46.8%, and 45.2%, respectively) compared to controls; other treatments had modest effects. EPA effects on cholesterol domain formation were dose-dependent. In membranes with 1:1 C/P (predisposing domain formation), DHA, but not EPA, dose-dependently increased membrane fluidity. DHA also induced cholesterol domain formation without affecting temperature-induced changes in-bilayer unit cell periodicity relative to controls (d-space; 57Å-55Å over 15-30°C). Together, these data suggest simultaneous formation of distinct cholesterol-rich ordered domains and cholesterol-poor disordered domains in the presence of DHA. By contrast, EPA had no effect on cholesterol domain formation and produced larger d-space values relative to controls (60Å-57Å; p<0.05) over the same temperature range, suggesting a more uniform maintenance of lipid dynamics despite the presence of cholesterol. These data indicate that EPA and DHA had different effects on membrane bilayer width, membrane fluidity, and cholesterol crystalline domain formation; suggesting omega-3 fatty acids with differing chain length or unsaturation may differentially influence membrane lipid dynamics and structural organization as a result of distinct phospholipid/sterol interactions. Copyright © 2016. Published by Elsevier B.V.

Structure and functions of simple membrane-water interfaces. [Abstract only

NASA Technical Reports Server (NTRS)

Pohorille, A.; Wilson, M. A.

1994-01-01

The structure and functions of the earliest ancestors of contemporary cells are focal points in studies of the origin of life. Probably the first cell-like structures were vesicles - closed, spheroidal structures with aqueous medium trapped inside. The membranous walls of vesicles were most likely bilayers composed of simple amphiphilic material available on early earth. The membrane studied was composed of glycerol 1-monooleate (GMO). Glycerol forms the polar head group and the oily tail contains 18 carbon atoms. All head groups have been found to be located in two narrow regions at the interfaces with water. The membrane interior, formed by the hydrophobic tails, is quite fluid with chain disorder increasing towards the center of the bilayer. These results are in agreement with x-ray and neutron scattering data from related bilayers. The width of the membrane is not constant, but fluctuates in time and space. Occasional thinning defects in the membrane, observed during the course of the simulations, may have a significant influence on rates of passive transport of small molecules across membranes. It has been found that water penetrates the head group region but not the oily interior of the membrane. Water molecules near the interface are oriented by dipoles of the head groups. The resulting electrostatic potential across the interface, determined in our simulations, has been found to be markedly larger than across the water-oil interface. This quantity has been implicated as the source of selectivity, with respect to the sign of the charge, as an ion approaches the interface and during transport of hydrophobic ions across membranes.

Effects of cholesterol on pore formation in lipid bilayers induced by human islet amyloid polypeptide fragments: A coarse-grained molecular dynamics study

NASA Astrophysics Data System (ADS)

Xu, Weixin; Wei, Guanghong; Su, Haibin; Nordenskiöld, Lars; Mu, Yuguang

2011-11-01

Disruption of the cellular membrane by the amyloidogenic peptide, islet amyloid polypeptide (IAPP), has been considered as one of the mechanisms of β-cell death during type 2 diabetes. The N-terminal region (residues 1-19) of the human version of IAPP is suggested to be primarily responsible for the membrane-disrupting effect of the full-length hIAPP peptide. However, the detailed assembly mode of hIAPP1-19 with membrane remains unclear. To gain insight into the interactions of hIAPP1-19 oligomer with the model membrane, we have employed coarse-grained molecular dynamics self-assembly simulations to study the aggregation of hIAPP1-19 fragments in the binary lipid made of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic dipalmitoylphosphatidylserine (DPPS) in the presence and absence of different levels of cholesterol content. The membrane-destabilizing effect of hIAPP1-19 is found to be modulated by the presence of cholesterol. In the absence of cholesterol, hIAPP1-19 aggregates prefer to locate inside the bilayer, forming pore-like assemblies. While in the presence of cholesterol molecules, the lipid bilayer becomes more ordered and stiff, and the hIAPP1-19 aggregates are dominantly positioned at the bilayer-water interface. The action of cholesterol may suggest a possible way to maintain the membrane integrity by small molecule interference.

Hybrid continuum-coarse-grained modeling of erythrocytes

NASA Astrophysics Data System (ADS)

Lyu, Jinming; Chen, Paul G.; Boedec, Gwenn; Leonetti, Marc; Jaeger, Marc

2018-06-01

The red blood cell (RBC) membrane is a composite structure, consisting of a phospholipid bilayer and an underlying membrane-associated cytoskeleton. Both continuum and particle-based coarse-grained RBC models make use of a set of vertices connected by edges to represent the RBC membrane, which can be seen as a triangular surface mesh for the former and a spring network for the latter. Here, we present a modeling approach combining an existing continuum vesicle model with a coarse-grained model for the cytoskeleton. Compared to other two-component approaches, our method relies on only one mesh, representing the cytoskeleton, whose velocity in the tangential direction of the membrane may be different from that of the lipid bilayer. The finitely extensible nonlinear elastic (FENE) spring force law in combination with a repulsive force defined as a power function (POW), called FENE-POW, is used to describe the elastic properties of the RBC membrane. The mechanical interaction between the lipid bilayer and the cytoskeleton is explicitly computed and incorporated into the vesicle model. Our model includes the fundamental mechanical properties of the RBC membrane, namely fluidity and bending rigidity of the lipid bilayer, and shear elasticity of the cytoskeleton while maintaining surface-area and volume conservation constraint. We present three simulation examples to demonstrate the effectiveness of this hybrid continuum-coarse-grained model for the study of RBCs in fluid flows.

Functional, Responsive Materials Assembled from Recombinant Oleosin

NASA Astrophysics Data System (ADS)

Hammer, Daniel

Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

The antiepileptic drug diphenylhydantoin affects the structure of the human erythrocyte membrane.

PubMed

Suwalsky, Mario; Mennickent, Sigrid; Norris, Beryl; Villena, Fernando; Cuevas, Francisco; Sotomayor, Carlos P

2004-01-01

Phenytoin (diphenylhydantoin) is an antiepileptic agent effective against all types of partial and tonic-clonic seizures. Phenytoin limits the repetitive firing of action potentials evoked by a sustained depolarization of mouse spinal cord neurons maintained in vitro. This effect is mediated by a slowing of the rate of recovery of voltage activated Na+ channels from inactivation. For this reasons it was thought of interest to study the binding affinities of phenytoin with cell membranes and their perturbing effects upon membrane structures. The effects of phenytoin on the human erythrocyte membrane and molecular models have been investigated in the present work. This report presents the following evidence that phenytoin interacts with cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that phenytoin perturbed a class of lipids found in the outer moiety of cell membranes; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the polar head groups and acyl chains of the erythrocyte membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes the formation of echinocytes was observed, due to the insertion of phenytoin in the outer monolayer of the red cell membrane. This is the first time that an effect of phenytoin on the red cell shape is described. However, the effects of the drug were observed at concentrations higher than those currently found in plasma when phenytoin is therapeutically administered.

Interactions and Translational Dynamics of Phosphatidylinositol Bisphosphate (PIP2) Lipids in Asymmetric Lipid Bilayers.

PubMed

Shi, Xiaojun; Kohram, Maryam; Zhuang, Xiaodong; Smith, Adam W

2016-02-23

Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.

HIV and Drug Resistance: Hitting a Moving Target | Center for Cancer Research

Cancer.gov

Prior research revealed how HIV-1 makes its destructive entry into the target cell by fusing together the cholesterol-rich lipid bilayer of the viral envelope—made with key glycoproteins gp120 and gp41—and the host cell’s plasma membrane. Cell-viral interactions begin with the binding of gp120 to the CD4 receptor molecule on the target cell, followed by gp120 binding to coreceptors. These coreceptors likely reside in structures called lipid rafts—areas in the cell plasma membrane that are rich in cholesterol, saturated fatty acids, and certain proteins that facilitate the entry of viruses into host cells. Finally, sequences in gp41 trigger the fusion of the viral and cellular lipid bilayers. The lipid rafts are then involved in the production of new viral particles.

Ordering of lipid membranes altered by boron nitride nanosheets.

PubMed

Zhang, Yonghui; Li, Zhen; Chan, Chun; Ma, Jiale; Zhi, Chunyi; Cheng, Xiaolin; Fan, Jun

2018-02-07

Boron nitride nanosheets are novel promising nanomaterials with a lower cytotoxicity than graphene making them a better candidate for biomedical applications. However, there is no systematic study on how they interact with cell membranes. Here we employed large scale all-atom molecular dynamics simulations to provide molecular details of the structure and properties of membranes after the insertion of boron nitride nanosheets. Our results reveal that the boron nitride nanosheet can extract phospholipids from the lipid bilayers and is enveloped by the membrane. Afterwards, the acyl chains of lipid molecules re-orient and become more ordered. As a result, a fluid to gel phase transition occurs in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer. Consequently, the bending moduli of the bilayers increase, and the diffusivity of the individual lipid molecule decreases. These changes will affect relevant cellular activities, such as endocytosis and signal transduction. Our study provides novel insights into the biocompatibility and cytotoxicity of boron nitride nanosheets, which may facilitate the design of safer nanocarriers, antibiotics and other bio-nanotechnology applications.

Nanosized CaP-silk fibroin-PCL-PEG-PCL/PCL based bilayer membranes for guided bone regeneration.

PubMed

Türkkan, Sibel; Pazarçeviren, A Engin; Keskin, Dilek; Machin, Nesrin E; Duygulu, Özgür; Tezcaner, Ayşen

2017-11-01

Guided bone regeneration (GBR) concept has been developed to prevent the formation of non-functional scar tissue layer on defect site by undertaking barrier role. In this study, a new bilayer membrane which consisted of one layer of electrospun silk fibroin/PCL-PEG-PCL incorporating nanocalcium phosphate (SPCA) 1 and one layer of PCL membrane was developed for GBR. To improve the osteoconductivity of membranes, nanosized calcium phosphate particles synthesized by Flame Spray Pyrolysis method were incorporated into membranes at 10% (wt) (SPCA10) and 20% (wt) (SPCA20) of the polymer content. The structural and chemical analyses revealed the well-integrated two layers of membranes with a total thickness of ca 100μm. In the regenerative layer, the highly porous mesh structure had a thickness of 12.6μm with randomly oriented fibers having diameters around 760nm, and nanoparticles dispersed homogenously. The mechanical test results showed remarkable improvement on the tensile strength of membranes with incorporation of nanoparticles. Higher water affinity of nanoCaP included membranes was proved by lower contact angle values and higher percent water uptake capacity. Biomineralization assay revealed that nucleation and growth of apatites around fibers of SPCA10 and SPCA20 were apparent while on SPCA0 apatite minerals were barely detected after 10days. Human dental pulp stem cells (DPSC) were seeded on electrospun layer of the bilayer membranes for biocompatibility and osteo-compatibility study. Increasing nanoCaP amount resulted in higher cell adhesion, proliferation, ALP activity and calcium deposition on membranes. These overall results confirmed the biocompatibility and potential applicability of proposed membranes for GBR treatments. Copyright © 2017 Elsevier B.V. All rights reserved.

Defining the free-energy landscape of curvature-inducing proteins on membrane bilayers.

PubMed

Tourdot, Richard W; Ramakrishnan, N; Radhakrishnan, Ravi

2014-08-01

Curvature-sensing and curvature-remodeling proteins, such as Amphiphysin, Epsin, and Exo70, are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors; they aggregate to membrane regions matching their intrinsic curvature; as well as induce curvature in cell membranes to stabilize emergent high curvature, nonspherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test particle (or field) insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membrane-in particular, we use this method to track the onset of morphological transitions in the membrane at elevated protein densities. We validate this approach by comparing the results from the Widom method with those of thermodynamic integration and Bennett acceptance ratio methods. Furthermore, the predictions from the Widom method have been tested against analytical calculations of the excess chemical potential at infinite dilution. Our results are useful in precisely quantifying the free-energy landscape, and also in determining the phase boundaries associated with curvature-induction, curvature-sensing, and morphological transitions. This approach can be extended to studies exploring the role of thermal fluctuations and other external (control) variables, such as membrane excess area, in shaping curvature-mediated interactions on bilayer membranes.

Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

PubMed

Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

2014-04-22

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

Force Generation by Membrane-Associated Myosin-I

PubMed Central

Pyrpassopoulos, Serapion; Arpağ, Göker; Feeser, Elizabeth A.; Shuman, Henry; Tüzel, Erkan; Ostap, E. Michael

2016-01-01

Vertebrate myosin-IC (Myo1c) is a type-1 myosin that links cell membranes to the cytoskeleton via its actin-binding motor domain and its phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding tail domain. While it is known that Myo1c bound to PtdIns(4,5)P2 in fluid-lipid bilayers can propel actin filaments in an unloaded motility assay, its ability to develop forces against external load on actin while bound to fluid bilayers has not been explored. Using optical tweezers, we measured the diffusion coefficient of single membrane-bound Myo1c molecules by force-relaxation experiments, and the ability of ensembles of membrane-bound Myo1c molecules to develop and sustain forces. To interpret our results, we developed a computational model that recapitulates the basic features of our experimental ensemble data and suggests that Myo1c ensembles can generate forces parallel to lipid bilayers, with larger forces achieved when the myosin works away from the plane of the membrane or when anchored to slowly diffusing regions. PMID:27156719

Alcohols Reduce Lateral Membrane Pressures: Predictions from Molecular Theory

PubMed Central

Frischknecht, Amalie L.; Frink, Laura J. Douglas

2006-01-01

We explore the effects of alcohols on fluid lipid bilayers using a molecular theory with a coarse-grained model. We show that the trends predicted from the theory in the changes in area per lipid, alcohol concentration in the bilayer, and area compressibility modulus, as a function of alcohol chain length and of the alcohol concentration in the solvent far from the bilayer, follow those found experimentally. We then use the theory to study the effect of added alcohol on the lateral pressure profile across the membrane, and find that added alcohol reduces the surface tensions at both the headgroup/solvent and headgroup/tailgroup interfaces, as well as the lateral pressures in the headgroup and tailgroup regions. These changes in lateral pressures could affect the conformations of membrane proteins, providing a nonspecific mechanism for the biological effects of alcohols on cells. PMID:16980354

Interaction of elaiophylin with model bilayer membrane

NASA Astrophysics Data System (ADS)

Genova, J.; Dencheva-Zarkova, M.

2017-01-01

Elaiophylin is a new macrodiolide antibiotic, which is produced by the Streptomyces strains [1]. It displays biological activities against Gram-positive bacteria and fungi. The mode of action of this antibiotic has been attributed to an alteration of the membrane permeability. When this antibiotic is inserted into the bilayer membranes destabilization of the membrane and formation of ion-penetrable channels is observed. The macrodiolide antibiotic forms stable cation selective ion channels in synthetic lipid bilayer membranes. The aim of this work was to study the interactions of Elaiophylin with model bilayer membranes and to get information on the mechanical properties of lipid bilayers in presence of this antibiotic. Patch-clamp technique [2] were used in the study

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.

Neutron diffraction studies of amphipathic helices in phospholipid bilayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradshaw, J.P.; Gilchrist, P.J.; Duff, K.C.

The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt tomore » investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.« less

Lipid Biomembrane in Ionic Liquids

NASA Astrophysics Data System (ADS)

Yoo, Brian; Jing, Benxin; Shah, Jindal; Maginn, Ed; Zhu, Y. Elaine; Department of Chemical and Biomolecular Engineering Team

2014-03-01

Ionic liquids (ILs) have been recently explored as new ``green'' chemicals in several chemical and biomedical processes. In our pursuit of understanding their toxicities towards aquatic and terrestrial organisms, we have examined the IL interaction with lipid bilayers as model cell membranes. Experimentally by fluorescence microscopy, we have directly observed the disruption of lipid bilayer by added ILs. Depending on the concentration, alkyl chain length, and anion hydrophobicity of ILs, the interaction of ILs with lipid bilayers leads to the formation of micelles, fibrils, and multi-lamellar vesicles for IL-lipid complexes. By MD computer simulations, we have confirmed the insertion of ILs into lipid bilayers to modify the spatial organization of lipids in the membrane. The combined experimental and simulation results correlate well with the bioassay results of IL-induced suppression in bacteria growth, thereby suggesting a possible mechanism behind the IL toxicity. National Science Foundation, Center for Research Computing at Notre Dame.

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; ...

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

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

Elicitation of anti-Sendai virus cytotoxic T lymphocytes by viral and H-2 antigens incorporated into the same lipid bilayer by membrane fusion and by reconstitution into liposomes.

PubMed

Hale, A H; Lyles, D S; Fan, D P

1980-02-01

We have investigated the minimal molecular requirements for elicitation of anti-Sendai virus cytotoxic T lymphocytes (CTL), and the minimal molecular requirements for the recognition and lysis processes associated with anti-Sendai virus CTL-target cell interactions. This report demonstrates a) that the hemagglutinin-neuraminidase and/or fusion glycoproteins of Sendai virus can elicit anti-Sendai virus CTL and b) that these glycoproteins and H-2 antigens must be within the same membrane lipid bilayer for effective elicitation of anti-Sendai-virus CTL and for effective recognition and lysis of target cells by anti-Sendai virus CTL.

Defining the free-energy landscape of curvature-inducing proteins on membrane bilayers

PubMed Central

Tourdot, Richard W.; Ramakrishnan, N.; Radhakrishnan, Ravi

2015-01-01

Curvature-sensing and curvature-remodeling proteins, such as Amphiphysin, Epsin, and Exo70, are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors; they aggregate to membrane regions matching their intrinsic curvature; as well as induce curvature in cell membranes to stabilize emergent high curvature, nonspherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test particle (or field) insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membrane—in particular, we use this method to track the onset of morphological transitions in the membrane at elevated protein densities. We validate this approach by comparing the results from the Widom method with those of thermodynamic integration and Bennett acceptance ratio methods. Furthermore, the predictions from the Widom method have been tested against analytical calculations of the excess chemical potential at infinite dilution. Our results are useful in precisely quantifying the free-energy landscape, and also in determining the phase boundaries associated with curvature-induction, curvature-sensing, and morphological transitions. This approach can be extended to studies exploring the role of thermal fluctuations and other external (control) variables, such as membrane excess area, in shaping curvature-mediated interactions on bilayer membranes. PMID:25215768

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 transmembrane proteins.

Subnanometer structure of an asymmetric model membrane: Interleaflet coupling influences domain properties

DOE PAGES

Heberle, Frederick A.; Marquardt, Drew; Doktorova, Milka; ...

2016-04-29

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order tomore » produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Lastly, our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.« less

Effects of Stretching Speed on Mechanical Rupture of Phospholipid/Cholesterol Bilayers: Molecular Dynamics Simulation

PubMed Central

Shigematsu, Taiki; Koshiyama, Kenichiro; Wada, Shigeo

2015-01-01

Rupture of biological cell membrane under mechanical stresses is critical for cell viability. It is triggered by local rearrangements of membrane molecules. We investigated the effects of stretching speed on mechanical rupture of phospholipid/cholesterol bilayers using unsteady molecular dynamics simulations. We focused on pore formation, the trigger of rupture, in a 40 mol% cholesterol-including bilayer. The unsteady stretching was modeled by proportional and temporal scaling of atom positions at stretching speeds from 0.025 to 30 m/s. The effects of the stretching speed on the critical areal strain, where the pore forms, is composed of two regimes. At low speeds (<1.0 m/s), the critical areal strain is insensitive to speed, whereas it significantly increases at higher speeds. Also, the strain is larger than that of a pure bilayer, regardless of the stretching speeds, which qualitatively agrees with available experimental data. Transient recovery of the cholesterol and phospholipid molecular orientations was evident at lower speeds, suggesting the formation of a stretch-induced interdigitated gel-like phase. However, this recovery was not confirmed at higher speeds or for the pure bilayer. The different responses of the molecular orientations may help explain the two regimes for the effect of stretching speed on pore formation. PMID:26471872

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 thosemore » 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.« less

Melittin-induced cholesterol reorganization in lipid bilayer membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Shuo; Heller, William T.

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 thosemore » 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.« less

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced sensitivity of a microfabricated resonator using a graphene-polystyrene bilayer membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Minhyuk; Lee, Eunho; Cho, Kilwon

2014-08-18

A graphene layer was synthesized using chemical vapor deposition methods and a polystyrene solution was spin-cast onto the graphene film. The graphene-polystyrene bilayer membrane was attached between the two tines of a microfabricated quartz tuning fork (QTF). The modulus of the graphene-polystyrene bilayer was measured to be twice that of a pristine polystyrene membrane. Exposure of the membrane-coated QTF to ethanol vapor decreased the resonance frequency of the microresonator. The bilayer membrane-coated QTF produced a frequency change that was three times the change obtained using a polystyrene membrane-coated QTF, with a lower degree of degradation in the Q factor. Themore » limit of detection of the bilayer membrane-coated QTF to ethanol vapor was determined to be 20 ppm.« less

Interaction of Local Anesthetics with Biomembranes Consisting of Phospholipids and Cholesterol: Mechanistic and Clinical Implications for Anesthetic and Cardiotoxic Effects

PubMed Central

2013-01-01

Despite a long history in medical and dental application, the molecular mechanism and precise site of action are still arguable for local anesthetics. Their effects are considered to be induced by acting on functional proteins, on membrane lipids, or on both. Local anesthetics primarily interact with sodium channels embedded in cell membranes to reduce the excitability of nerve cells and cardiomyocytes or produce a malfunction of the cardiovascular system. However, the membrane protein-interacting theory cannot explain all of the pharmacological and toxicological features of local anesthetics. The administered drug molecules must diffuse through the lipid barriers of nerve sheaths and penetrate into or across the lipid bilayers of cell membranes to reach the acting site on transmembrane proteins. Amphiphilic local anesthetics interact hydrophobically and electrostatically with lipid bilayers and modify their physicochemical property, with the direct inhibition of membrane functions, and with the resultant alteration of the membrane lipid environments surrounding transmembrane proteins and the subsequent protein conformational change, leading to the inhibition of channel functions. We review recent studies on the interaction of local anesthetics with biomembranes consisting of phospholipids and cholesterol. Understanding the membrane interactivity of local anesthetics would provide novel insights into their anesthetic and cardiotoxic effects. PMID:24174934

Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane.

PubMed

Williams, Dumaine; Vicôgne, Jérome; Zaitseva, Irina; McLaughlin, Stuart; Pessin, Jeffrey E

2009-12-01

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

Neutron Reflectivity as a Tool for Physics-Based Studies of Model Bacterial Membranes.

PubMed

Barker, Robert D; McKinley, Laura E; Titmuss, Simon

2016-01-01

The principles of neutron reflectivity and its application as a tool to provide structural information at the (sub-) molecular unit length scale from models for bacterial membranes are described. The model membranes can take the form of a monolayer for a single leaflet spread at the air/water interface, or bilayers of increasing complexity at the solid/liquid interface. Solid-supported bilayers constrain the bilayer to 2D but can be used to characterize interactions with antimicrobial peptides and benchmark high throughput lab-based techniques. Floating bilayers allow for membrane fluctuations, making the phase behaviour more representative of native membranes. Bilayers of varying levels of compositional accuracy can now be constructed, facilitating studies with aims that range from characterizing the fundamental physical interactions, through to the characterization of accurate mimetics for the inner and outer membranes of Gram-negative bacteria. Studies of the interactions of antimicrobial peptides with monolayer and bilayer models for the inner and outer membranes have revealed information about the molecular control of the outer membrane permeability, and the mode of interaction of antimicrobials with both inner and outer membranes.

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

PubMed Central

Metkar, Sunil S.; Wang, Baikun; Catalan, Elena; Anderluh, Gregor; Gilbert, Robert J. C.; Pardo, Julian; Froelich, Christopher J.

2011-01-01

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells. PMID:21931672

Prevention of intra-abdominal adhesion by bi-layer electrospun membrane.

PubMed

Jiang, Shichao; Wang, Wei; Yan, Hede; Fan, Cunyi

2013-06-04

The aim of this study was to compare the anti-adhesion efficacy of a bi-layer electrospun fibrous membrane consisting of hyaluronic acid-loaded poly(ε-caprolactone) (PCL) fibrous membrane as the inner layer and PCL fibrous membrane as the outer layer with a single-layer PCL electrospun fibrous membrane in a rat cecum abrasion model. The rat model utilized a cecal abrasion and abdominal wall insult surgical protocol. The bi-layer and PCL membranes were applied between the cecum and the abdominal wall, respectively. Control animals did not receive any treatment. After postoperative day 14, a visual semiquantitative grading scale was used to grade the extent of adhesion. Histological analysis was performed to reveal the features of adhesion tissues. Bi-layer membrane treated animals showed significantly lower adhesion scores than control animals (p < 0.05) and a lower adhesion score compared with the PCL membrane. Histological analysis of the bi-layer membrane treated rat rarely demonstrated tissue adhesion while that of the PCL membrane treated rat and control rat showed loose and dense adhesion tissues, respectively. Bi-layer membrane can efficiently prevent adhesion formation in abdominal cavity and showed a significantly decreased adhesion tissue formation compared with the control.

Small-molecule photostabilizing agents are modifiers of lipid bilayer properties.

PubMed

Alejo, Jose L; Blanchard, Scott C; Andersen, Olaf S

2013-06-04

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 membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives.

PubMed

Jiang, Yao-Wen; Guo, Hao-Yue; Chen, Zhan; Yu, Zhi-Wu; Wang, Zhifei; Wu, Fu-Gen

2016-07-05

Lipid rafts are highly ordered small microdomains mainly composed of glycosphingolipids, cholesterol, and protein receptors. Optically distinguishing lipid raft domains in cell membranes would greatly facilitate the investigations on the structure and dynamics of raft-related cellular behaviors, such as signal transduction, membrane transport (endocytosis), adhesion, and motility. However, current strategies about the visualization of lipid raft domains usually suffer from the low biocompatibility of the probes, invasive detection, or ex situ observation. At the same time, naturally derived biomacromolecules have been extensively used in biomedical field and their interaction with cells remains a long-standing topic since it is closely related to various fundamental studies and potential applications. Herein, noninvasive visualization of lipid raft domains in model lipid bilayers (supported lipid bilayers and giant unilamellar vesicles) and live cells was successfully realized in situ using fluorescent biomacromolecules: the fluorescein isothiocyanate (FITC)-labeled glycol chitosan molecules. We found that the lipid raft domains in model or real membranes could be specifically stained by the FITC-labeled glycol chitosan molecules, which could be attributed to the electrostatic attractive interaction and/or hydrophobic interaction between the probes and the lipid raft domains. Since the FITC-labeled glycol chitosan molecules do not need to completely insert into the lipid bilayer and will not disturb the organization of lipids, they can more accurately visualize the raft domains as compared with other fluorescent dyes that need to be premixed with the various lipid molecules prior to the fabrication of model membranes. Furthermore, the FITC-labeled glycol chitosan molecules were found to be able to resist cellular internalization and could successfully visualize rafts in live cells. The present work provides a new way to achieve the imaging of lipid rafts and also sheds new light on the interaction between biomacromolecules and lipid membranes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Chi-Yuan; Kausik, Ravinath; Han, Songi, E-mail: songi@chem.ucsb.edu

Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed {sup 1}H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5–10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroupsmore » or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster surface water diffusivity. Another is the concurrent tightening of lipid packing that reduces passive, possibly unwanted, diffusion of ions and water across the bilayer.« less

Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

PubMed

Discher, D E; Mohandas, N

1996-10-01

Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton.

Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties

PubMed Central

Montal, M.; Mueller, P.

1972-01-01

Bimolecular membranes are formed from two lipid monolayers at an air-water interface by the apposition of their hydrocarbon chains when an aperture in a Teflon partition separating two aqueous phases is lowered through the interface. Formation of the membrane is monitored by an increase of the electrical capacity, as measured with a voltage clamp. Electrical resistance of the unmodified membrane is analogous to that of conventional planar bilayers (black lipid membranes) prepared in the presence of a hydrocarbon solvent, i.e., 106-108 ohm cm2; the resistance can be lowered to values of 103 ohm cm2 by gramicidin, an antibiotic that modifies the conductance only when the membranes are of biomolecular thickness. In contrast to the resistance, there is a significant difference between the capacity of bilayers made from mono-layers and that of hydrocarbon-containing bilayers made by phase transition; the average values are 0.9 and 0.45 μF cm-2, respectively. The value of 0.9 μF cm-2 approximates that of biological membranes. Assuming a dielectric constant of 2.1 for the hydrocarbon region, the dielectric thickness, as calculated from a capacity of 0.9 μF cm-2, is 22 Å. This value is 6-10 Å smaller than the actual thickness of the hydrocarbon region of bilayers and cell membranes, as determined by x-ray diffraction. The difference may be due to a limited penetration of water into the hydrocarbon region near the ester groups that would lower the electrical resistance of this region and reduce the dielectric thickness. Asymmetric membranes have been formed by adjoining two lipid monolayers of different chemical composition. Images PMID:4509315

Sonoporation at Small and Large Length Scales: Effect of Cavitation Bubble Collapse on Membranes.

PubMed

Fu, Haohao; Comer, Jeffrey; Cai, Wensheng; Chipot, Christophe

2015-02-05

Ultrasound has emerged as a promising means to effect controlled delivery of therapeutic agents through cell membranes. One possible mechanism that explains the enhanced permeability of lipid bilayers is the fast contraction of cavitation bubbles produced on the membrane surface, thereby generating large impulses, which, in turn, enhance the permeability of the bilayer to small molecules. In the present contribution, we investigate the collapse of bubbles of different diameters, using atomistic and coarse-grained molecular dynamics simulations to calculate the force exerted on the membrane. The total impulse can be computed rigorously in numerical simulations, revealing a superlinear dependence of the impulse on the radius of the bubble. The collapse affects the structure of a nearby immobilized membrane, and leads to partial membrane invagination and increased water permeation. The results of the present study are envisioned to help optimize the use of ultrasound, notably for the delivery of drugs.

Slurry spin coating of thin film yttria stabilized zirconia/gadolinia doped ceria bi-layer electrolytes for solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Kim, Hyun Joong; Kim, Manjin; Neoh, Ke Chean; Han, Gwon Deok; Bae, Kiho; Shin, Jong Mok; Kim, Gyu-Tae; Shim, Joon Hyung

2016-09-01

Thin ceramic bi-layered membrane comprising yttria-stabilized zirconia (YSZ) and gadolinia-doped ceria (GDC) is fabricated by the cost-effective slurry spin coating technique, and it is evaluated as an electrolyte of solid oxide fuel cells (SOFCs). It is demonstrated that the slurry spin coating method is capable of fabricating porous ceramic films by adjusting the content of ethyl-cellulose binders in the source slurry. The porous GDC layer deposited by spin coating under an optimal condition functions satisfactorily as a cathode-electrolyte interlayer in the test SOFC stack. A 2-μm-thick electrolyte membrane of the spin-coated YSZ/GDC bi-layer is successfully deposited as a dense and stable film directly on a porous NiO-YSZ anode support without any interlayers, and the SOFC produces power output over 200 mW cm-2 at 600 °C, with an open circuit voltage close to 1 V. Electrochemical impedance spectra analysis is conducted to evaluate the performance of the fuel cell components in relation with the microstructure of the spin-coated layers.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

PubMed Central

Vecchiarelli, Anthony G.; Li, Min; Mizuuchi, Michiyo; Hwang, Ling Chin; Seol, Yeonee; Neuman, Keir C.; Mizuuchi, Kiyoshi

2016-01-01

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified “burst” patterns—radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator. PMID:26884160

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.

PubMed

Vecchiarelli, Anthony G; Li, Min; Mizuuchi, Michiyo; Hwang, Ling Chin; Seol, Yeonee; Neuman, Keir C; Mizuuchi, Kiyoshi

2016-03-15

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified "burst" patterns--radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.

Contact bubble bilayers with flush drainage.

PubMed

Iwamoto, Masayuki; Oiki, Shigetoshi

2015-03-16

Planar lipid bilayers have been used to form stable bilayers into which membrane proteins are reconstituted for measurements of their function under an applied membrane potential. Recently, a lipid bilayer membrane is formed by the apposition of two monolayers that line an oil-electrolyte interface. Here, a bilayer membrane system is developed with picoliter bubbles under mechanically and chemically manipulable conditions. A water bubble lined with a phospholipid monolayer is blown from a glass pipette into an oil phase. Two blowing pipettes are manipulated, and bubbles (each with a diameter of ~ 50 μm) are held side by side to form a bilayer, which is termed a contact bubble bilayer. With the electrode implemented in the blowing pipette, currents through the bilayer are readily measured. The intra-bubble pressure is varied with the pressure-controller, leading to various sizes of the bubble and the membrane area. A rapid solution exchange system is developed by introducing additional pressure-driven injection pipettes, and the blowing pipette works as a drain. The solution is exchanged within 20 ms. Also, an asymmetric membrane with different lipid composition of each leaflet is readily formed. Example applications of this versatile method are presented to characterize the function of ion channels.

Contact Bubble Bilayers with Flush Drainage

PubMed Central

Iwamoto, Masayuki; Oiki, Shigetoshi

2015-01-01

Planar lipid bilayers have been used to form stable bilayers into which membrane proteins are reconstituted for measurements of their function under an applied membrane potential. Recently, a lipid bilayer membrane is formed by the apposition of two monolayers that line an oil-electrolyte interface. Here, a bilayer membrane system is developed with picoliter bubbles under mechanically and chemically manipulable conditions. A water bubble lined with a phospholipid monolayer is blown from a glass pipette into an oil phase. Two blowing pipettes are manipulated, and bubbles (each with a diameter of ~ 50 μm) are held side by side to form a bilayer, which is termed a contact bubble bilayer. With the electrode implemented in the blowing pipette, currents through the bilayer are readily measured. The intra-bubble pressure is varied with the pressure-controller, leading to various sizes of the bubble and the membrane area. A rapid solution exchange system is developed by introducing additional pressure-driven injection pipettes, and the blowing pipette works as a drain. The solution is exchanged within 20 ms. Also, an asymmetric membrane with different lipid composition of each leaflet is readily formed. Example applications of this versatile method are presented to characterize the function of ion channels. PMID:25772819

Amide I SFG Spectral Line Width Probes the Lipid-Peptide and Peptide-Peptide Interactions at Cell Membrane In Situ and in Real Time.

PubMed

Zhang, Baixiong; Tan, Junjun; Li, Chuanzhao; Zhang, Jiahui; Ye, Shuji

2018-06-13

The balance of lipid-peptide and peptide-peptide interactions at cell membrane is essential to a large variety of cellular processes. In this study, we have experimentally demonstrated for the first time that sum frequency generation vibrational spectroscopy can be used to probe the peptide-peptide and lipid-peptide interactions in cell membrane in situ and in real time by determination of the line width of amide I band of protein backbone. Using a "benchmark" model of α-helical WALP23, it is found that the dominated lipid-peptide interaction causes a narrow line width of the amide I band, whereas the peptide-peptide interaction can markedly broaden the line width. When WALP23 molecules insert into the lipid bilayer, a quite narrow line width of the amide I band is observed because of the lipid-peptide interaction. In contrast, when the peptide lies down on the bilayer surface, the line width of amide I band becomes very broad owing to the peptide-peptide interaction. In terms of the real-time change in the line width, the transition from peptide-peptide interaction to lipid-peptide interaction is monitored during the insertion of WALP23 into 1,2-dipalmitoyl- sn-glycero-3-phospho-(1'- rac-glycerol) (DPPG) lipid bilayer. The dephasing time of a pure α-helical WALP23 in 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-(1'- rac-glycerol) and DPPG bilayer is determined to be 2.2 and 0.64 ps, respectively. The peptide-peptide interaction can largely accelerate the dephasing time.

Shallow boomerang-shaped influenza hemagglutinin G13A mutant structure promotes leaky membrane fusion.

PubMed

Lai, Alex L; Tamm, Lukas K

2010-11-26

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation.

Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion*

PubMed Central

Lai, Alex L.; Tamm, Lukas K.

2010-01-01

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation. PMID:20826788

Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities

USDA-ARS?s Scientific Manuscript database

Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavin...

Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

PubMed

Martins, Danubia Batista; Nasário, Fábio Domingues; Silva-Gonçalves, Laiz Costa; de Oliveira Tiera, Vera Aparecida; Arcisio-Miranda, Manoel; Tiera, Marcio José; Dos Santos Cabrera, Marcia Perez

2018-02-01

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport.

PubMed

Hong, Seungpyo; Bielinska, Anna U; Mecke, Almut; Keszler, Balazs; Beals, James L; Shi, Xiangyang; Balogh, Lajos; Orr, Bradford G; Baker, James R; Banaszak Holl, Mark M

2004-01-01

We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.

Evaluation of the ability of arsenic species to traverse cell membranes by simple diffusion using octanol-water and liposome-water partition coefficients.

PubMed

Chávez-Capilla, Teresa; Maher, William; Kelly, Tamsin; Foster, Simon

2016-11-01

Arsenic metabolism in living organisms is dependent on the ability of different arsenic species to traverse biological membranes. Simple diffusion provides an alternative influx and efflux route to mediated transport mechanisms that can increase the amount of arsenic available for metabolism in cells. Using octanol-water and liposome-water partition coefficients, the ability of arsenous acid, arsenate, methylarsonate, dimethylarsinate, thio-methylarsonate, thio-dimethylarsinic acid, arsenotriglutathione and monomethylarsonic diglutathione to diffuse through the lipid bilayer of cell membranes was investigated. Molecular modelling of arsenic species was used to explain the results. All arsenic species with the exception of arsenate, methylarsonate and thio-methylarsonate were able to diffuse through the lipid bilayer of liposomes, with liposome-water partition coefficients between 0.04 and 0.13. Trivalent arsenic species and thio-pentavalent arsenic species showed higher partition coefficients, suggesting that they can easily traverse cell membranes by passive simple diffusion. Given the higher toxicity of these species compared to oxo-pentavalent arsenic species, this study provides evidence supporting the risk associated with human exposure to trivalent and thio-arsenic species. Copyright © 2016. Published by Elsevier B.V.

Viral membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

Interaction of Boron Nitride Nanosheets with Model Cell Membranes.

PubMed

Hilder, Tamsyn A; Gaston, Nicola

2016-06-03

Boron nitride nanomaterials have attracted attention for biomedical applications, due to their improved biocompatibility when compared with carbon nanomaterials. Recently, graphene and graphene oxide nanosheets have been shown, both experimentally and computationally, to destructively extract phospholipids from Escherichia coli. Boron nitride nanosheets (BNNSs) have exciting potential biological and environmental applications, for example the ability to remove oil from water. These applications are likely to increase the exposure of prokaryotes and eukaryotes to BNNSs. Yet, despite their promise, the interaction between BNNSs and cell membranes has not yet been investigated. Here, all-atom molecular dynamics simulations were used to demonstrate that BNNSs are spontaneously attracted to the polar headgroups of the lipid bilayer. The BNNSs do not passively cross the lipid bilayer, most likely due to the large forces experienced by the BNNSs. This study provides insight into the interaction of BNNSs with cell membranes and may aid our understanding of their improved biocompatibility. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HDL particles incorporate into lipid bilayers - a combined AFM and single molecule fluorescence microscopy study.

PubMed

Plochberger, Birgit; Röhrl, Clemens; Preiner, Johannes; Rankl, Christian; Brameshuber, Mario; Madl, Josef; Bittman, Robert; Ros, Robert; Sezgin, Erdinc; Eggeling, Christian; Hinterdorfer, Peter; Stangl, Herbert; Schütz, Gerhard J

2017-11-21

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) - a main carrier of cholesterol in the blood stream - to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.

El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes.

PubMed

Ikigai, H; Ono, T; Iwata, M; Nakae, T; Shimamura, T

1997-05-15

We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers. The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent. The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation. The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes. These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis.

Influence of ester-modified lipids on bilayer structure.

PubMed

Villanueva, Diana Y; Lim, Joseph B; Klauda, Jeffery B

2013-11-19

Lipid membranes function as barriers for cells to prevent unwanted chemicals from entering the cell and wanted chemicals from leaving. Because of their hydrophobic interior, membranes do not allow water to penetrate beyond the headgroup region. We performed molecular simulations to examine the effects of ester-modified lipids, which contain ester groups along their hydrocarbon chains, on bilayer structure. We chose two lipids from those presented in Menger et al. [J. Am. Chem. Soc. 2006, 128, 14034] with ester groups in (1) the upper half of the lipid chain (MEPC) and (2) the middle and end of the lipid chain (MGPC). MGPC (30%)/POPC bilayers formed stable water pores of diameter 5-7 Å, but MGPC (22%)/POPC and MEPC (30%)/POPC bilayers did not form these defects. These pores were similar to those formed during electroporation; i.e., the head groups lined the pore and allowed water and ions to transport across the bilayer. However, we found that lateral organization of the MGPC lipids into clusters, instead of an electric field or charge disparity as in electroporation, was essential for pore formation. On the basis of this, we propose an overall mechanism for pore formation. The similarities between the ester-modified lipids and byproducts of lipid peroxidation with multiple hydrophilic groups in the middle of the chain suggest that free radical reactions with unsaturated lipids and sterols result in fundamental changes that may be similar to what is seen in bilayers with ester-modified lipids.

Creation and Relaxation of Phospholipid Compositional Asymmetry in Lipid Bilayers Examined by Sum-Frequency Vibrational Spectroscopy

NASA Astrophysics Data System (ADS)

Anglin, Timothy C.; Brown, Krystal; Conboy, John C.

2010-08-01

Eukaryotic cells contain an asymmetric distribution of phospholipids in the two leaflets of the lipid bilayer which is known to contribute to cellular function. In the plasma membrane of eukaryotic cells, the aminophospholipids with phosphatidylserine (PS) and phosphatidylethanolamine (PE) headgroups are predominately located on the cytosolic leaflet while sphingolipids with phosphatidylcholine (PC) headgroups and sphingomeylin are on the extra-cellular leaflet. There have been a number of theories about the mechanism of transbilayer movement of lipids in cellular systems and the physical process by which lipid compositional asymmetry in the plasma membrane of eukaryotic cells is maintained. It is generally accepted that a significant barrier to native lipid translocation (movement between leaflets of the bilayer) exists which is related to the energetic penalty of moving the hydrophilic headgroup of a phospholipid through the hydrophobic core of the membrane. Overcoming this energetic barrier represents the rate limiting step in the spontaneous flip-flop of phospholipids in biological membranes, yet, while numerous kinetic studies of phospholipid flip-flop have been conducted, few researchers have reported thermodynamic parameters for the process. Using methods of classical surface chemistry coupled with nonlinear optical methods, we have developed a novel analytical approach, using sum-frequency vibrational spectroscopy (SFVS), to selectively probe lipid compositional asymmetry in a planar supported lipid bilayer. This new method allows for the detection of lipid flip-flop kinetics and compositional asymmetry without the need for a fluorescent or spin-labeled lipid species by exploiting the coherent nature of SFVS. The SFVS intensity arising from the terminal methyl groups of the lipid fatty acid chains is used as an internal probe to directly monitor the compositional asymmetry in planar supported lipid bilayers (PSLBs(. By selectively deuterating a sub-population of lipids, the SFVS intensity is proportional to the population difference between hydrogenated lipids in the top, NT, and the bottom, NB, leaflets due to the cancellation of the SFVS signal arising from lipids hydrogenated residing in an anti-parallel arrangement, allowing us to directly relate the measured intensity to the population difference in the bilayer (Equation 1) and provides a direct measure of the percent asymmetry (%AS) in the membrane (Equation 2). ICH3∝(NT-NB)2 (1) %AS = (NT-NB)/NTotal×100 (2) In this presentation, the effect of lipid composition, headgroup and fatty acid chemical structure, on the rate and thermodynamics of lipid transbilayer migration and the electrostatic induction of lipid asymmetry will be discussed.

Mechanical properties of electrospun bilayer fibrous membranes as potential scaffolds for tissue engineering.

PubMed

Pu, Juan; Komvopoulos, Kyriakos

2014-06-01

Bilayer fibrous membranes of poly(l-lactic acid) (PLLA) were fabricated by electrospinning, using a parallel-disk mandrel configuration that resulted in the sequential deposition of a layer with fibers aligned across the two parallel disks and a layer with randomly oriented fibers, both layers deposited in a single process step. Membrane structure and fiber alignment were characterized by scanning electron microscopy and two-dimensional fast Fourier transform. Because of the intricacies of the generated electric field, bilayer membranes exhibited higher porosity than single-layer membranes consisting of randomly oriented fibers fabricated with a solid-drum collector. However, despite their higher porosity, bilayer membranes demonstrated generally higher elastic modulus, yield strength and toughness than single-layer membranes with random fibers. Bilayer membrane deformation at relatively high strain rates comprised multiple abrupt microfracture events characterized by discontinuous fiber breakage. Bilayer membrane elongation yielded excessive necking of the layer with random fibers and remarkable fiber stretching (on the order of 400%) in the layer with fibers aligned in the stress direction. In addition, fibers in both layers exhibited multiple localized necking, attributed to the nonuniform distribution of crystalline phases in the fibrillar structure. The high membrane porosity, good mechanical properties, and good biocompatibility and biodegradability of PLLA (demonstrated in previous studies) make the present bilayer membranes good scaffold candidates for a wide range of tissue engineering applications. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Probing the interaction between nanoparticles and lipid membranes by quartz crystal microbalance with dissipation monitoring

NASA Astrophysics Data System (ADS)

Yousefi, Nariman; Tufenkji, Nathalie

2016-12-01

There is increasing interest in using quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the interaction of nanoparticles (NPs) with model surfaces. The high sensitivity, ease of use and the ability to monitor interactions in real-time has made it a popular technique for colloid chemists, biologists, bioengineers and biophysicists. QCM-D has been recently used to probe the interaction of NPs with supported lipid bilayers (SLBs) as model cell membranes. The interaction of NPs with SLBs is highly influenced by the quality of the lipid bilayers. Unlike many surface sensitive techniques, using QCM-D, the quality of SLBs can be assessed in real-time, hence QCM-D studies on SLB-NP interactions are less prone to the artefacts arising from bilayers that are not well formed. The ease of use and commercial availability of a wide range of sensor surfaces also have made QCM-D a versatile tool for studying NP interactions with lipid bilayers. In this review, we summarize the state-of-the-art on QCM-D based techniques for probing the interactions of NPs with lipid bilayers.

Exploring the interactions between peptides and lipid bilayers using coherent anti-Stokes Raman scattering and two-photon fluorescence

NASA Astrophysics Data System (ADS)

Mari, M.; Mouras, R.; Downes, A.; Elfick, A.

2011-06-01

We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging on which we combine Coherent Anti-Stokes Raman Scattering (CARS) with Two Photon Excitation Fluorescence (TPEF) using a Nd: YVO4 pump laser. We acquired 2PEF, CARS, and phase contrast images of Multilamellar Vesicles (MLVs) and Giant Unilamellar Vesicles (GUVs), as well as Raman spectra of the constituent lipids. A wide range of peptides are harmful to cells by altering the structure of the biological membranes. This effect depends on the composition of the membrane and the chemical structure of the peptide. The peptide we studied is the beta amyloid Aβ which is a major component of the amyloid plaques deposited on neuronal membranes of Alzheimer's disease (AD) patients. AD is neurodegenerative disorder in which the hallmark symptoms include cognitive decline and dementia[2] and is characterized by the formation of extracellular amyloid fibrils on the neuronal membranes of the brain. Many questions still remain unanswered concerning the destabilization of cellular ionic homeostasis due to pores formed during the interactions of lipid membranes with peptides. In this project, biomimics of cell membranes are used. The structures that best mimic the plasma membranes are MLVs or GUVs. These vesicles are formed using the gentle hydration technique[3] or the electroformation technique[4] respectively and are composed of phospholipids such as DOPC, DPPC, D62PPC and their binary mixtures. The MLVs and GUVs imaging by CARS and TPEF microscopy not only permits the direct imaging of the leakage phenomenon caused by the toxic peptide (Aβ) on the lipid bilayer, but also records simultaneously the lateral structure of the bilayer and peptide distribution in the plane across the membrane.

MD simulation study of direct permeation of a nanoparticle across the cell membrane under an external electric field.

PubMed

Shimizu, Kenta; Nakamura, Hideya; Watano, Satoru

2016-06-09

Nanoparticles (NPs) have been attracting much attention for biomedical and pharmaceutical applications. In most of the applications, NPs are required to translocate across the cell membrane and to reach the cell cytosol. Experimental studies have reported that by applying an electric field NPs can directly permeate across the cell membrane without the confinement of NPs by endocytic vesicles. However, damage to the cell can often be a concern. Understanding of the mechanism underlying the direct permeation of NPs under an external electric field can greatly contribute to the realization of a technology for the direct delivery of NPs. Here we investigated the permeation of a cationic gold NP across a phospholipid bilayer under an external electric field using a coarse-grained molecular dynamics simulation. When an external electric field that is equal to the membrane breakdown intensity was applied, a typical NP delivery by electroporation was shown: the cationic gold NP directly permeated across a lipid bilayer without membrane wrapping of the NP, while a persistent transmembrane pore was formed. However, when a specific range of the electric field that is lower than the membrane breakdown intensity was applied, a unique permeation pathway was exhibited: the generated transmembrane pore immediately resealed after the direct permeation of NP. Furthermore, we found that the affinity of the NP for the membrane surface is a key for the self-resealing of the pore. Our finding suggests that by applying an electric field in a suitable range NPs can be directly delivered into the cell with less cellular damage.

Rheology of Membrane-Attached Minimal Actin Cortices.

PubMed

Nöding, Helen; Schön, Markus; Reinermann, Corinna; Dörrer, Nils; Kürschner, Aileen; Geil, Burkhard; Mey, Ingo; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

2018-04-26

The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P 2 ) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P 2 /ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G 0 to the node density of the MAC.

Lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes

DOE PAGES

Smith, Jeremy C.; Cheng, Xiaolin; Nickels, Jonathan D.

2015-07-29

Understanding of cell membrane organization has evolved significantly from the classic fluid mosaic model. It is now recognized that biological membranes are highly organized structures, with differences in lipid compositions between inner and outer leaflets and in lateral structures within the bilayer plane, known as lipid rafts. These organizing principles are important for protein localization and function as well as cellular signaling. However, the mechanisms and biophysical basis of lipid raft formation, structure, dynamics and function are not clearly understood. One key question, which we focus on in this review, is how lateral organization and leaflet compositional asymmetry are coupled.more » Detailed information elucidating this question has been sparse because of the small size and transient nature of rafts and the experimental challenges in constructing asymmetric bilayers. Resolving this mystery will require advances in both experimentation and modeling. We discuss here the preparation of model systems along with experimental and computational approaches that have been applied in efforts to address this key question in membrane biology. Furthermore, we seek to place recent and future advances in experimental and computational techniques in context, providing insight into in-plane and transverse organization of biological membranes.« less

Study of pH (low) insertion peptides (pHLIPs) interaction with lipid bilayer of membrane

NASA Astrophysics Data System (ADS)

Weerakkody, Dhammika

The pH-dependent interactions of pHLIPsRTM (pH (Low) Insertion Peptides) with lipid bilayer of membrane provides an opportunity to study and address fundamental questions of protein folding/insertion into membrane and unfolding/exit, as well as develop novel approach to target acidic diseased tissue such as cancer, ischemic myocardium, infection and others. The main goal of the work presented here is to answer the following questions: - What is the molecular mechanism of spontaneous insertion and folding of a peptide in a lipid bilayer of membrane; - What is the molecular mechanism of unfolding and exit of a peptide from a lipid bilayer of membrane; - How polar cargo attached to a peptide's inserting end might affect the process of insertion into a lipid bilayer of membrane; How sequence variation will affect a peptide's interactions with a lipid bilayer of membrane (partitioning into bilayer at neutral and low pH; apparent pK of insertion) with the main goal to identify the best pHLIP variants for imaging and therapy of pathological states such as cancer and others. It has been demonstrated that pHLIP insertion into a membrane is associated with the protonation of Asp/Glu residues, which leads to an increase of hydrophobicity that triggers the folding and insertion of the peptide across a lipid bilayer. The insertion of the pHLIP is unidirectional and it is accompanied by the release of energy. Therefore, the energy of membrane associated-folding can be used to favor the movement of cell-impermeable polar cargo molecules across the hydrophobic membrane bilayer when they are attached to the inserting end of pHLIP. Both pH-targeting behavior and molecular translocation have been demonstrated in cultured cells and in vivo. Thus, there is an opportunity to develop a novel concept in drug delivery, which is based on the use of a monomeric, pH-sensitive peptide molecular transporter, to deliver agents that are significantly more polar than conventional drugs. Understanding the molecular events that occur when a peptide inserts across a membrane, folds, or exits from it and unfolds provides crucial information for the development of new drug delivery agents, as well as improving our understanding of the first step of membrane-associate protein folding. The promise of exploiting tumor acidosis as a cancer biomarker has not been fully realized in clinical practice, even though the acidity has been a known property since the work of Otto Warburg nearly a century ago. The problem has been to find a practical way to target acidity. pHLIP reversibly folds and inserts across membranes in response to pH changes, and this discovery has led to a novel way to target acidic tissue. Steady state biophysical studies have revealed the molecular mechanism of pHLIP action, which is based on the increase of hydrophobicity of carboxyl groups when they become protonated under mildly acidic conditions, leading to peptide insertion into a membrane. It has been shown that pHLIP can target acidic tissue and selectively translocate polar, cell-impermeable molecules across membranes in response to low extracellular pH. As noted in the Molecular Imaging and Contrast Agent Database (MICAD) at NCBI, a pHLIP labeled with a fluorescent dye, or a PET- and SPECT- agents (64Cu-DOTA, 18F, 99Tc) is a marker for in vivo acidity. All prior studies in vivo were carried out with the WT-pHLIP sequence and showed that a good contrast and tumor to blood ratio can be achieved only more than 24 hours after pHLIP injection, when it has accumulated in the tumor and largely cleared from the blood. However, for the use of pHLIP-based radioactive imaging agents in the clinic, a more rapid background signal reduction is absolutely essential. We have conducted research in order to address this important need, to tune tumor targeting properties, and to broaden our understanding of the molecular mechanism of pHLIP action. A family of 16 pHLIP variants has been designed based on chemical and physical principles and comprehensive biophysical studies were performed with non-labeled peptides. We have successfully established a set of design criteria and identified the pHLIP candidates for imaging and therapeutic applications, including lead compounds for PET/SPECT and fluorescence/MR imaging.

Membrane Interactions of hIAPP Monomer and Oligomer with Lipid Membranes by Molecular Dynamics Simulations.

PubMed

Zhang, Mingzhen; Ren, Baiping; Liu, Yonglan; Liang, Guizhao; Sun, Yan; Xu, Lijian; Zheng, Jie

2017-08-16

Interaction of human islet amyloid polypeptide (hIAPP) peptides with cell membrane is crucial for the understanding of amyloid toxicity associated with Type II diabetes (T2D). While it is known that the hIAPP-membrane interactions are considered to promote hIAPP aggregation into fibrils and induce membrane disruption, the membrane-induced conformation, orientation, aggregation, and adsorption behaviors of hIAPP peptides have not been well understood at the atomic level. Herein, we perform all-atom explicit-water molecular dynamics (MD) simulations to study the adsorption, orientation, and surface interaction of hIAPP aggregates with different sizes (monomer to tetramer) and conformations (monomer with α-helix and tetramer with β-sheet-rich U-turn) upon adsorption on the lipid bilayers composed of both pure zwitterionic POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and mixed anionic POPC/POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (3:1) lipids. MD simulation results show that hIAPP monomer with α-helical conformation and hIAPP pentamer with β-sheet conformation can adsorb on both POPC and POPC/POPE bilayers via a preferential orientation of N-terminal residues facing toward the bilayer surface. The hIAPP aggregates show stronger interactions with mixed POPC/POPE lipids than pure POPC lipids, consistent with experimental observation that hIAPP adsorption and fibrililation are enhanced on mixed lipid bilayers. While electrostatic interactions are main attractive forces to drive the hIAPP aggregates to adsorb on both bilayers, the introduction of the more hydrophilic head groups of POPE lipids further promote the formation of the interfacial hydrogen bonds. Complement to our previous studies of hIAPP aggregates in bulk solution, this computational work increases our knowledge about the mechanism of amyloid peptide-membrane interactions that is central to the understanding the progression of all amyloid diseases.

HIV gp41 fusion peptide increases membrane ordering in a cholesterol-dependent fashion.

PubMed

Lai, Alex L; Freed, Jack H

2014-01-07

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Interaction between the NS4B amphipathic helix, AH2, and charged lipid headgroups alters membrane morphology and AH2 oligomeric state--Implications for the Hepatitis C virus life cycle.

PubMed

Ashworth Briggs, Esther L; Gomes, Rafael G B; Elhussein, Malaz; Collier, William; Findlow, I Stuart; Khalid, Syma; McCormick, Chris J; Williamson, Philip T F

2015-08-01

The non-structural protein 4B (NS4B) from Hepatitis C virus (HCV) plays a pivotal role in the remodelling of the host cell's membranes, required for the formation of the viral replication complex where genome synthesis occurs. NS4B is an integral membrane protein that possesses a number of domains vital for viral replication. Structural and biophysical studies have revealed that one of these, the second amphipathic N-terminal helix (AH2), plays a key role in these remodelling events. However, there is still limited understanding of the mechanism through which AH2 promotes these changes. Here we report on solid-state NMR and molecular dynamics studies that demonstrate that AH2 promotes the clustering of negatively charged lipids within the bilayer, a process that reduces the strain within the bilayer facilitating the remodelling of the lipid bilayer. Furthermore, the presence of negatively charged lipids within the bilayer appears to promote the disassociation of AH2 oligomers, highlighting a potential role for lipid recruitment in regulating NS protein interactions. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Amphipathic amines affect membrane excitability in paramecium: role for bilayer couple.

PubMed Central

Browning, J L; Nelson, D L

1976-01-01

Amphipathic amines and local anesthetics stimulated reversal of the ciliary beating direction in wild-type Paramecium. Ca++ influx across the surface membrane and the consequent increase in internal Ca++ causes ciliary reversal and backward swimming. Mutant cells of the "Pawn" class, which lack a "gating" mechanism for regulating Ca++ influx, did not swim backwards in the presence of local anesthetics. Local anesthetics stimulated the passive efflux of K+ but had no effect on the active transport of K+ or Ca++. Apparently passive influx of Ca++ also was stimulated by local anesthetics as evidenced by their effects on swimming direction. These data can be interpreted in terms of the "bilayer couple" hypothesis of Sheetz and Singer [(1974) Proc. Nat. Acad. Sci. USA 71, 4457-4461]: amphipathic drugs affect cells by asymmetric insertion into one face of the lipid bilayer. As predicted by this hypothesis, the drugs' effects were seen only after a short time lag, and quaternary amines were less effective than tertiary amines. The effect on behavior was caused by any of several amphipathic cations, and the relative potency was a function of their hydrophobicity. Amphipathic anions, which according to the hypothesis would insert into the opposite face of the lipid bilayer, had little effect on ciliary reversal. Asymmetric perturbation of the lipid bilayer with amphipathic cations may trigger the opening of the Ca++ gate. Images PMID:1061147

Antimicrobial Peptide Simulations and the Influence of Force Field on the Free Energy for Pore Formation in Lipid Bilayers.

PubMed

Bennett, W F Drew; Hong, Chun Kit; Wang, Yi; Tieleman, D Peter

2016-09-13

Due to antimicrobial resistance, the development of new drugs to combat bacterial and fungal infections is an important area of research. Nature uses short, charged, and amphipathic peptides for antimicrobial defense, many of which disrupt the lipid membrane in addition to other possible targets inside the cell. Computer simulations have revealed atomistic details for the interactions of antimicrobial peptides and cell-penetrating peptides with lipid bilayers. Strong interactions between the polar interface and the charged peptides can induce bilayer deformations - including membrane rupture and peptide stabilization of a hydrophilic pore. Here, we performed microsecond-long simulations of the antimicrobial peptide CM15 in a POPC bilayer expecting to observe pore formation (based on previous molecular dynamics simulations). We show that caution is needed when interpreting results of equilibrium peptide-membrane simulations, given the length of time single trajectories can dwell in local energy minima for 100's of ns to microseconds. While we did record significant membrane perturbations from the CM15 peptide, pores were not observed. We explain this discrepancy by computing the free energy for pore formation with different force fields. Our results show a large difference in the free energy barrier (ca. 40 kJ/mol) against pore formation predicted by the different force fields that would result in orders of magnitude differences in the simulation time required to observe spontaneous pore formation. This explains why previous simulations using the Berger lipid parameters reported pores induced by charged peptides, while with CHARMM based models pores were not observed in our long time-scale simulations. We reconcile some of the differences in the distance dependent free energies by shifting the free energy profiles to account for thickness differences between force fields. The shifted curves show that all the models describe small defects in lipid bilayers in a consistent manner, suggesting a common physical basis.

Decoupling of bilayer leaflets under gas supersaturation: nitrogen nanobubbles in a membrane and their implication in decompression sickness

NASA Astrophysics Data System (ADS)

Li, Jing; Zhang, Xianren; Cao, Dapeng

2018-05-01

Decompression sickness (also known as diver’s sickness) is a disease that arises from the formation of a bubble inside the body caused by rapid decompression from high atmospheric pressures. However, the nature of pre-existing micronuclei that are proposed for interpreting the formation and growth of the bubble, as well as their very existence, is still highly controversial. In this work, atomistic molecular dynamics simulations are employed to investigate the nucleation of gas bubbles under the condition of nitrogen supersaturation, in the presence of a lipid bilayer and lipid micelle representing other macromolecules with a smaller hydrophobic region. Our simulation results demonstrate that by crossing a small energy barrier, excess nitrogen molecules can enter the lipid bilayer nearly spontaneously, for which the hydrophobic core serves as a potential well for gas enrichment. At a rather low nitrogen supersaturation, gas molecules in the membrane are dispersed in the hydrophobic region of the bilayer, with a slight increase in membrane thickness. But as the level of gas supersaturation reaches a threshold, the accumulation of N2 molecules in the bilayer center causes the two leaflets to be decoupled and the formation of nanobubbles. Therefore, we propose a nucleation mechanism for bubble formation in a supersaturated solution of inert gas: a cell membrane acts as a potential well for gas enrichment, being an ideal location for forming nanobubbles that induce membrane damage at a high level of gas supersaturation. As opposed to previous models, the new mechanism involves forming gas nuclei in a very low-tension hydrophobic environment, and thus a rather low energy barrier is required and pre-existing bubble micronuclei are not needed.

Exploiting lipopolysaccharide-induced deformation of lipid bilayers to modify membrane composition and generate two-dimensional geometric membrane array patterns

DOE PAGES

Adams, Peter G.; Swingle, Kirstie L.; Paxton, Walter F.; ...

2015-05-27

Supported lipid bilayers have proven effective as model membranes for investigating biophysical processes and in development of sensor and array technologies. The ability to modify lipid bilayers after their formation and in situ could greatly advance membrane technologies, but is difficult via current state-of-the-art technologies. Here we demonstrate a novel method that allows the controlled post-formation processing and modification of complex supported lipid bilayer arrangements, under aqueous conditions. We exploit the destabilization effect of lipopolysaccharide, an amphiphilic biomolecule, interacting with lipid bilayers to generate voids that can be backfilled to introduce desired membrane components. We further demonstrate that when usedmore » in combination with a single, traditional soft lithography process, it is possible to generate hierarchically-organized membrane domains and microscale 2-D array patterns of domains. Significantly, this technique can be used to repeatedly modify membranes allowing iterative control over membrane composition. This approach expands our toolkit for functional membrane design, with potential applications for enhanced materials templating, biosensing and investigating lipid-membrane processes.« less

Effect of biocompatible polymers on the structural integrity of lipid bilayers under external stimuli

NASA Astrophysics Data System (ADS)

Wang, Jia-Yu; Kausik, Ravinath; Chen, Chi-Yuan; Han, Song-I.; Marks, Jeremy; Lee, Ka Yee

2010-03-01

Cell membrane dysfunction due to loss of structural integrity is the pathology of tissue death in trauma and common diseases. It is now established that certain biocompatible polymers, such as Poloxamer 188, Poloxamine 1107 and polyethylene glycol (PEG), are effective in sealing of injured cell membranes, and able to prevent acute necrosis. Despite these broad applications of these polymers for human health, the fundamental mechanisms by which these polymers interact with cell membranes are still under debate. Here, the effects of a group of biocompatible polymers on phospholipid membrane integrity under osmotic and oxidative stress were explored using giant unilamellar vesicles as model cell membranes. Our results suggest that the adsorption of the polymers on the membrane surface is responsible for the cell membrane resealing process due to its capability of slowing down the surface hydration dynamics.

Formation of 3D cholesterol crystals from 2D nucleation sites in lipid bilayer membranes: implications for atherosclerosis.

PubMed

Varsano, Neta; Fargion, Iael; Wolf, Sharon G; Leiserowitz, Leslie; Addadi, Lia

2015-02-04

Atherosclerosis is the major precursor of cardiovascular disease. The formation of cholesterol crystals in atherosclerotic plaques is associated with the onset of acute pathology. The cholesterol crystals induce physical injury in the plaque core, promoting cell apoptosis and triggering an increased inflammatory response. Herein we address the question of how cholesterol crystal formation occurs in atherosclerosis. We demonstrate that three-dimensional (3D) cholesterol crystals can undergo directed nucleation from bilayer membranes containing two-dimensional (2D) cholesterol crystalline domains. We studied crystal formation on supported lipid bilayers loaded with exogenous cholesterol and labeled using a monoclonal antibody that specifically recognizes ordered cholesterol arrays. Our findings show that 3D crystals are formed exclusively on the bilayer regions where there are segregated 2D cholesterol crystalline domains and that they form on the domains. This study has potentially significant implications for our understanding of the crucial step in the mechanism by which atherosclerotic lesions form.

Biomimetic polyesters and their role in ion transport across cell membranes.

PubMed

Jedliński, Z; Kurcok, P; Adamus, G; Juzwa, M

2000-01-01

Syntheses of biomimetic low-molecular weight poly-(R)-3-hydroxybutanoate mediated by three types of supramolecular catalysts are presented. The utility of these synthetic polyesters for preparation of artificial channels in phospholipid bilayers capable of sodium and calcium ion transport across cell membranes, is discussed. Further studies on possible applications of these bio-polymers for manufacturing drugs of prolonged activity are under way.

Interaction of hematoporphyrin with lipid membranes.

PubMed

Stępniewski, Michał; Kepczynski, Mariusz; Jamróz, Dorota; Nowakowska, Maria; Rissanen, Sami; Vattulainen, Ilpo; Róg, Tomasz

2012-04-26

Natural or synthetic porphyrins are being used as photosensitizers in photodiagnosis (PD) and photodynamic therapy (PDT) of malignancies and some other diseases. Understanding the interactions between porphyrins and cell membranes is therefore important to rationalize the uptake of photosensitizers and their passive transport through cell membranes. In this study, we consider the properties of hematoporphyrin (Hp), a well-known photosensitizer for PD and PDT, in the presence of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer that we use as a model system for protein-free cell membranes. For this purpose, we employed 200 ns atomic-scale molecular dynamics (MD) simulations for five systems containing the neutral (Hp(0)) or the dianionic form (Hp(2-)) of Hp and the POPC bilayer. MD simulations allowed one to estimate the position, orientation, and dynamics of Hp molecules inside the membrane. The dye molecules were found to reside in the phospholipid headgroup area close to the carbonyl groups of the POPC acyl chains. Their orientations were dependent on the protonation state of two propionic groups. Hp(2-) was found to have a lower affinity to enter the membrane than the neutral form. The dianions, being in the aqueous phase, formed stable dimers with a strictly determined geometry. Our results fully supported the experimental data and provide a more detailed molecular-level description of the interactions of photosensitizers with lipid membranes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boreyko, Jonathan B; Mruetusatorn, Prachya; Sarles, Stephen A

Droplet interface bilayers (DIBs) are a robust platform for studying synthetic cellular membranes; however, to date no DIBs have been produced at cellular length scales. Here, we create microscale droplet interface bilayers ( DIBs) at the interface between aqueous femtoliter-volume droplets within an oil-filled microfluidic channel. The uniquely large area-to-volume ratio of the droplets results in strong evaporation effects, causing the system to transition through three distinct regimes. First, the two adjacent droplets shrink into the shape of a single spherical droplet, where an augmented lipid bilayer partitions two hemi-spherical volumes. In the second regime, the combined effects of themore » shrinking monolayers and growing bilayer force the confined bilayer to buckle to conserve its mass. Finally, at a bending moment corresponding to a critical shear stress, the buckling bilayer fissions a vesicle to regulate its shape and stress. The DIBs produced here enable evaporation-induced bilayer dynamics reminiscent of endo- and exocytosis in cells.« less

Quantifying the Relationship between Curvature and Electric Potential in Lipid Bilayers.

PubMed

Bruhn, Dennis S; Lomholt, Michael A; Khandelia, Himanshu

2016-06-02

Cellular membranes mediate vital cellular processes by being subject to curvature and transmembrane electrical potentials. Here we build upon the existing theory for flexoelectricity in liquid crystals to quantify the coupling between lipid bilayer curvature and membrane potentials. Using molecular dynamics simulations, we show that headgroup dipole moments, the lateral pressure profile across the bilayer, and spontaneous curvature all systematically change with increasing membrane potentials. In particular, there is a linear dependence between the bending moment (the product of bending rigidity and spontaneous curvature) and the applied membrane potentials. We show that biologically relevant membrane potentials can induce biologically relevant curvatures corresponding to radii of around 500 nm. The implications of flexoelectricity in lipid bilayers are thus likely to be of considerable consequence both in biology and in model lipid bilayer systems.

Enhancement of Cell Membrane Invaginations, Vesiculation and Uptake of Macromolecules by Protonation of the Cell Surface

PubMed Central

Ben-Dov, Nadav; Korenstein, Rafi

2012-01-01

The different pathways of endocytosis share an initial step involving local inward curvature of the cell’s lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell’s surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake. PMID:22558127

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heller, William T.; Rai, Durgesh K.

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

Effect of intrinsic curvature and edge tension on the stability of binary mixed-membrane three-junctions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Jasmine M.; Abrams, Cameron F.; Deserno, Markus

We use a combination of coarse-grained molecular dynamics simulations and theoretical modeling to examine three-junctions in mixed lipid bilayer membranes. These junctions are localized defect lines in which three bilayers merge in such a way that each bilayer shares one monolayer with one of the other two bilayers. The resulting local morphology is non-lamellar, resembling the threefold symmetric defect lines in inverse hexagonal phases, but it regularly occurs during membrane fission and fusion events. We realize a system of junctions by setting up a honeycomb lattice, which in its primitive cell contains two hexagons and four three-line junctions, permitting usmore » to study their stability as well as their line tension. We specifically consider the effects of lipid composition and intrinsic curvature in binary mixtures, which contain a fraction of negatively curved lipids in a curvature-neutral background phase. Three-junction stability results from a competition between the junction and an open edge, which arises if one of the three bilayers detaches from the other two. We show that the stable phase is the one with the lower defect line tension. The strong and opposite monolayer curvatures present in junctions and edges enhance the mole fraction of negatively curved lipids in junctions and deplete it in edges. This lipid sorting affects the two line tensions and in turn the relative stability of the two phases. It also leads to a subtle entropic barrier for the transition between junction and edge that is absent in uniform membranes.« less

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

DOE PAGES

Heller, William T.; Rai, Durgesh K.

2017-01-16

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

Interaction of Aspirin (Acetylsalicylic Acid) with Lipid Membranes

PubMed Central

Barrett, Matthew A.; Zheng, Songbo; Roshankar, Golnaz; Alsop, Richard J.; Belanger, Randy K. R.; Huynh, Chris; Kučerka, Norbert; Rheinstädter, Maikel C.

2012-01-01

We studied the interaction of Aspirin (acetylsalicylic acid) with lipid membranes using x-ray diffraction for bilayers containing up to 50 mol% of aspirin. From 2D x-ray intensity maps that cover large areas of reciprocal space we determined the position of the ASA molecules in the phospholipid bilayers and the molecular arrangement of the molecules in the plane of the membranes. We present direct experimental evidence that ASA molecules participate in saturated lipid bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and preferably reside in the head group region of the membrane. Up to 50 mol% ASA molecules can be dissolved in this type of bilayer before the lateral membrane organization is disturbed and the membranes are found to form an ordered, 2D crystal-like structure. Furthermore, ASA and cholesterol were found to co-exist in saturated lipid bilayers, with the ASA molecules residing in the head group region and the cholesterol molecules participating in the hydrophobic membrane core. PMID:22529913

Non-Brownian diffusion in lipid membranes: Experiments and simulations.

PubMed

Metzler, R; Jeon, J-H; Cherstvy, A G

2016-10-01

The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane-binding particles. We discuss how membrane compartmentalisation and the particle-membrane binding energy may impact the dynamics and response of lipid membranes. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Lipopolysaccharide Membrane Building and Simulation

PubMed Central

Jo, Sunhwan; Wu, Emilia L.; Stuhlsatz, Danielle; Klauda, Jeffery B.; Widmalm, Göran; Im, Wonpil

2015-01-01

Summary While membrane simulations are widely employed to study the structure and dynamics of various lipid bilayers and membrane proteins in the bilayers, simulations of lipopolysaccharides (LPS) in membrane environments have been limited due to its structural complexity, difficulties in building LPS-membrane systems, and lack of appropriate molecular force field. In this work, as a first step to extend CHARMM-GUI Membrane Builder to incorporate LPS molecules and to explore their structures and dynamics in membrane environments using molecular dynamics simulations, we describe step-by-step procedures to build LPS bilayer systems using CHARMM and the recently developed CHARMM carbohydrate and lipid force fields. Such procedures are illustrated by building various bilayers of Escherichia coli O6 LPS and their preliminary simulation results are given in terms of per-LPS area and density distributions of various components along the membrane normal. PMID:25753722

Sodium selective ion channel formation in living cell membranes by polyamidoamine dendrimer.

PubMed

Nyitrai, Gabriella; Keszthelyi, Tamás; Bóta, Attila; Simon, Agnes; Tőke, Orsolya; Horváth, Gergő; Pál, Ildikó; Kardos, Julianna; Héja, László

2013-08-01

Polyamidoamine (PAMAM) dendrimers are highly charged hyperbranched protein-like polymers that are known to interact with cell membranes. In order to disclose the mechanisms of dendrimer-membrane interaction, we monitored the effect of PAMAM generation five (G5) dendrimer on the membrane permeability of living neuronal cells followed by exploring the underlying structural changes with infrared-visible sum frequency vibrational spectroscopy (SVFS), small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). G5 dendrimers were demonstrated to irreversibly increase the membrane permeability of neurons that could be blocked in low-[Na(+)], but not in low-[Ca(2+)] media suggesting the formation of specific Na(+) permeable channels. SFVS measurements on silica supported DPPG-DPPC bilayers suggested G5-specific trans-polarization of the membrane. SAXS data and freeze-fracture TEM imaging of self-organized DPPC vesicle systems demonstrated disruption of DPPC vesicle layers by G5 through polar interactions between G5 terminal amino groups and the anionic head groups of DPPC. We propose a nanoscale mechanism by which G5 incorporates into the membrane through multiple polar interactions that disrupt proximate membrane bilayer and shape a unique hydrophilic Na(+) ion permeable channel around the dendrimer. In addition, we tested whether these artificial Na(+) channels can be exploited as antibiotic tools. We showed that G5 quickly arrest the growth of resistant bacterial strains below 10μg/ml concentration, while they show no detrimental effect on red blood cell viability, offering the chance for the development of new generation anti-resistant antibiotics. Copyright © 2013 Elsevier B.V. All rights reserved.

The role of polyglutamine expansion and protein context in disease-related huntingtin/lipid interactions

NASA Astrophysics Data System (ADS)

Burke, Kathleen Anne

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 amino acids on the N-terminus (N17) and the polyproline domain on the C-terminal side of the polyQ domain have been shown to further modulate the aggregation process. Additionally, N17 appears to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-length (35Q, 46Q, 51Q, and myc- 53Q) or synthetic peptides with different polyQ domain flanking sequences (KK-Q35-KK, KK-Q 35-P10-KK, N17-Q35-KK, and N 17-Q35-P10-KK) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. By adding N-terminal tags to the htt exon1 fragments, the interaction with the lipid bilayer was impeded. The KK-Q35-KK and KK-Q 35-P10-KK peptides had no appreciable interaction with lipid bilayers. Interestingly, polyQ peptides with the N17 flanking sequence interacted with the bilayer. N17-Q35-KK formed discrete aggregates on the bilayer, but there was minimal membrane disruption. The N17-Q35-P10-KK peptide interacted more aggressively with the lipid bilayer in a manner reminiscent of the htt exon1 proteins.

Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes

PubMed Central

Ryu, Hyunil; Lee, Hwankyu; Iwata, Seigo; Choi, Sangbaek; Ki Kim, Moon; Kim, Young-Rok; Maruta, Shinsaku; Min Kim, Sun; Jeon, Tae-Joon

2015-01-01

Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations. PMID:26189604

Visualization of membrane RNAs

PubMed Central

JANAS, TADEUSZ; YARUS, MICHAEL

2003-01-01

Using fluorescence microscopy, we show that previously isolated membrane-binding RNAs coat artificial phospholipid membranes relatively uniformly, except for a frequent tendency to concentrate at bends, membrane junctions, and other unusual sites. Membrane RNAs can also be visualized as single molecules or isolated complexes by atomic force microscopy (AFM) of free RNAs on mica. Finally, RNAs can be seen within membranes by AFM of RNA-liposomes immobilized on hydrophobic mica surfaces. Monomer RNAs appear globular, as expected for small RNAs. When mixed under conditions in which RNAs bind bilayers, RNA 9 and RNA 10 combine to yield about 80% of RNAs as mainly linear oligomers of ≈2–8 molecules. Once inserted in membranes, the RNAs oligomerize further, yielding larger, irregular ropelike structures that prefer the edges of altered lipid patches. These properties can be interpreted in terms of RNA–RNA loop interactions, and the RNA effects on membranes can be explained in terms of an RNA preference for irregular lipid conformations. The RNA-bilayer system poses new opportunities for combining the properties of membranes and RNA in contemporary cells, and also in the ribocytes of an RNA world. PMID:14561885

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

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.

Modeling the effect of dynamic surfaces on membrane penetration

NASA Astrophysics Data System (ADS)

van Lehn, Reid; Alexander-Katz, Alfredo

2011-03-01

The development of nanoscale materials for targeted drug delivery is an important current pursuit in materials science. One task of drug carriers is to release therapeutic agents within cells by bypassing the cell membrane to maximize the effectiveness of their payload and minimize bodily exposure. In this work, we use coarse-grained simulations to study nanoparticles (NPs) grafted with hydrophobic and hydrophilic ligands that rearrange in response to the amphiphilic lipid bilayer. We demonstrate that this dynamic surface permits the NP to spontaneously penetrate to the bilayer midplane when the surface ligands are near an order-disorder transition. We believe that this work will lead to the design of new drug carriers capable of non-specifically accessing cell interiors based solely on their dynamic surface properties. Our work is motivated by existing nanoscale systems such as micelles, or NPs grafted with highly mobile ligands or polymer brushes.

Interactions of the baicalin and baicalein with bilayer lipid membranes investigated by cyclic voltammetry and UV-Vis spectroscopy.

PubMed

Zhang, Ying; Wang, Xuejing; Wang, Lei; Yu, Miao; Han, Xiaojun

2014-02-01

The baicalin and baicalein are the major flavonoids found in Radix Scutellariae, an essential herb in traditional Chinese medicine for thousands of years. The interactions of the baicalin and baicalein with lipid bilayer membranes were studied using cyclic voltammetry and UV-Vis spectroscopy. The thickness d of supported bilayer lipid membranes was calculated as d=4.59(±0.36) nm using AC impedance spectroscopy. The baicalein interacted with egg PC bilayer membranes in a dose-dependent manner. The responses of K3Fe(CN)6 on lipid bilayer membrane modified Pt electrode linearly increased in a concentration range of baicalein from 6.25μM to 25μM with a detection limit of 0.1μM and current-concentration sensitivity of 0.11(±0.01) μA/μM, and then reached a plateau from 25μM to 50μM. However the baicalin showed much weaker interactions with egg PC bilayer membranes. UV-Vis spectroscopy also confirmed that the baicalein could interact with egg PC membranes noticeably, but the interaction of baicalin with membranes was hard to be detected. The results provide useful information on understanding the mechanism of action of Radix Scutellariae in vivo. © 2013.

Membrane composition and dynamics: a target of bioactive virgin olive oil constituents.

PubMed

Lopez, Sergio; Bermudez, Beatriz; Montserrat-de la Paz, Sergio; Jaramillo, Sara; Varela, Lourdes M; Ortega-Gomez, Almudena; Abia, Rocio; Muriana, Francisco J G

2014-06-01

The endogenous synthesis of lipids, which requires suitable dietary raw materials, is critical for the formation of membrane bilayers. In eukaryotic cells, phospholipids are the predominant membrane lipids and consist of hydrophobic acyl chains attached to a hydrophilic head group. The relative balance between saturated, monounsaturated, and polyunsaturated acyl chains is required for the organization and normal function of membranes. Virgin olive oil is the richest natural dietary source of the monounsaturated lipid oleic acid and is one of the key components of the healthy Mediterranean diet. Virgin olive oil also contains a unique constellation of many other lipophilic and amphipathic constituents whose health benefits are still being discovered. The focus of this review is the latest evidence regarding the impact of oleic acid and the minor constituents of virgin olive oil on the arrangement and behavior of lipid bilayers. We highlight the relevance of these interactions to the potential use of virgin olive oil in preserving the functional properties of membranes to maintain health and in modulating membrane functions that can be altered in several pathologies. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

Physiologically-Relevant Modes of Membrane Interactions by the Human Antimicrobial Peptide, LL-37, Revealed by SFG Experiments

PubMed Central

Ding, Bei; Soblosky, Lauren; Nguyen, Khoi; Geng, Junqing; Yu, Xinglong; Ramamoorthy, Ayyalusamy; Chen, Zhan

2013-01-01

Antimicrobial peptides (AMPs) could become the next generation antibiotic compounds which can overcome bacterial resistance by disrupting cell membranes and it is essential to determine the factors underlying its mechanism of action. Although high-resolution NMR and other biological studies have provided valuable insights, it has been a major challenge to follow the AMP-membrane interactions at physiologically-relevant low peptide concentrations. In this study, we demonstrate a novel approach to overcome this major limitation by performing Sum Frequency Generation (SFG) vibrational spectroscopic experiments on lipid bilayers containing an AMP, LL-37. Our results demonstrate the power of SFG to study non-linear helical peptides and also infer that lipid-peptide interaction and the peptide orientation depend on the lipid membrane composition. The observed SFG signal changes capture the aggregating process of LL-37 on membrane. In addition, our SFG results on cholesterol-containing lipid bilayers indicate the inhibition effect of cholesterol on peptide-induced membrane permeation process. PMID:23676762

Physiologically-Relevant Modes of Membrane Interactions by the Human Antimicrobial Peptide, LL-37, Revealed by SFG Experiments

NASA Astrophysics Data System (ADS)

Ding, Bei; Soblosky, Lauren; Nguyen, Khoi; Geng, Junqing; Yu, Xinglong; Ramamoorthy, Ayyalusamy; Chen, Zhan

2013-05-01

Antimicrobial peptides (AMPs) could become the next generation antibiotic compounds which can overcome bacterial resistance by disrupting cell membranes and it is essential to determine the factors underlying its mechanism of action. Although high-resolution NMR and other biological studies have provided valuable insights, it has been a major challenge to follow the AMP-membrane interactions at physiologically-relevant low peptide concentrations. In this study, we demonstrate a novel approach to overcome this major limitation by performing Sum Frequency Generation (SFG) vibrational spectroscopic experiments on lipid bilayers containing an AMP, LL-37. Our results demonstrate the power of SFG to study non-linear helical peptides and also infer that lipid-peptide interaction and the peptide orientation depend on the lipid membrane composition. The observed SFG signal changes capture the aggregating process of LL-37 on membrane. In addition, our SFG results on cholesterol-containing lipid bilayers indicate the inhibition effect of cholesterol on peptide-induced membrane permeation process.

Physiologically-relevant modes of membrane interactions by the human antimicrobial peptide, LL-37, revealed by SFG experiments.

PubMed

Ding, Bei; Soblosky, Lauren; Nguyen, Khoi; Geng, Junqing; Yu, Xinglong; Ramamoorthy, Ayyalusamy; Chen, Zhan

2013-01-01

Antimicrobial peptides (AMPs) could become the next generation antibiotic compounds which can overcome bacterial resistance by disrupting cell membranes and it is essential to determine the factors underlying its mechanism of action. Although high-resolution NMR and other biological studies have provided valuable insights, it has been a major challenge to follow the AMP-membrane interactions at physiologically-relevant low peptide concentrations. In this study, we demonstrate a novel approach to overcome this major limitation by performing Sum Frequency Generation (SFG) vibrational spectroscopic experiments on lipid bilayers containing an AMP, LL-37. Our results demonstrate the power of SFG to study non-linear helical peptides and also infer that lipid-peptide interaction and the peptide orientation depend on the lipid membrane composition. The observed SFG signal changes capture the aggregating process of LL-37 on membrane. In addition, our SFG results on cholesterol-containing lipid bilayers indicate the inhibition effect of cholesterol on peptide-induced membrane permeation process.

Membrane alternatives in worlds without oxygen: Creation of an azotosome.

PubMed

Stevenson, James; Lunine, Jonathan; Clancy, Paulette

2015-02-01

The lipid bilayer membrane, which is the foundation of life on Earth, is not viable outside of biology based on liquid water. This fact has caused astronomers who seek conditions suitable for life to search for exoplanets within the "habitable zone," the narrow band in which liquid water can exist. However, can cell membranes be created and function at temperatures far below those at which water is a liquid? We take a step toward answering this question by proposing a new type of membrane, composed of small organic nitrogen compounds, that is capable of forming and functioning in liquid methane at cryogenic temperatures. Using molecular simulations, we demonstrate that these membranes in cryogenic solvent have an elasticity equal to that of lipid bilayers in water at room temperature. As a proof of concept, we also demonstrate that stable cryogenic membranes could arise from compounds observed in the atmosphere of Saturn's moon, Titan, known for the existence of seas of liquid methane on its surface.

New insights into the roles of proteins and lipids in membrane transport of fatty acids.

PubMed

Hamilton, James A

2007-01-01

Recent calculations of the apparent permeability coefficients for long-chain fatty acids (LCFA) in phospholipid bilayers provide a new perspective on their transport in a membrane. LCFA have permeabilities that are many orders of magnitude higher than glucose, amino acids, and ions. Transport of LCFA through membranes must therefore be considered to be much different from these nutrients, and there is no a priori requirement for catalysis by a membrane protein. New evidence indicates that the plasma membrane proteins postulated as catalysts for transporting LCFA into the cell fall into three categories. Some act as enzymes, mainly for the activation of LCFA to the acyl CoA, which is required for subsequent intracellular metabolism of LCFA. Other proteins appear to participate in sequestering and trafficking of LCFA. Finally, some proteins have undefined mechanisms. The established mechanisms are consistent with biophysical properties of LCFA in membranes, including fast free diffusion by "flip-flop" in the phospholipid bilayer.

Heterogeneous electrolyte (YSZ-Al 2O 3) based direct oxidation solid oxide fuel cell

NASA Astrophysics Data System (ADS)

Thokchom, J. S.; Xiao, H.; Rottmayer, M.; Reitz, T. L.; Kumar, B.

Bilayers comprised of dense and porous YSZ-Al 2O 3 (20 wt%) composite were tape cast, processed, and then fabricated into working solid oxide fuel cells (SOFCs). The porous part of the bilayer was converted into anode for direct oxidation of fuels by infiltrating CeO 2 and Cu. The cathode side of the bilayer was coated with an interlayer [YSZ-Al 2O 3 (20 wt%)]: LSM (1:1) and LSM as cathode. Several button cells were evaluated under hydrogen/air and propane/air atmospheres in intermediate temperature range and their performance data were analyzed. For the first time the feasibility of using YSZ-Al 2O 3 material for fabricating working SOFCs with high open circuit voltage (OCV) and power density is demonstrated. AC impedance spectroscopy and scanning electron microscopy (SEM) techniques were used to characterize the membrane and cell.

Using a patterned grating structure to create lipid bilayer platforms insensitive to air bubbles.

PubMed

Han, Chung-Ta; Chao, Ling

2015-01-07

Supported lipid bilayers (SLBs) have been used for various biosensing applications. The bilayer structure enables embedded lipid membrane species to maintain their native orientation, and the two-dimensional fluidity is crucial for numerous biomolecular interactions to occur. The platform integrated with a microfluidic device for reagent transport and exchange has great potential to be applied with surface analytical tools. However, SLBs can easily be destroyed by air bubbles during assay reagent transport and exchange. Here, we created a patterned obstacle grating structured surface in a microfluidic channel to protect SLBs from being destroyed by air bubbles. Unlike all of the previous approaches using chemical modification or adding protection layers to strengthen lipid bilayers, the uniqueness of this approach is that it uses the patterned obstacles to physically trap water above the bilayers to prevent the air-water interface from directly coming into contact with and peeling the bilayers. We showed that our platform with certain grating geometry criteria can provide promising protection to SLBs from air bubbles. The required obstacle distance was found to decrease when we increased the air-bubble movement speed. In addition, the interaction assay results from streptavidin and biotinylated lipids in the confined SLBs suggested that receptors at the SLBs retained the interaction ability after air-bubble treatment. The results showed that the developed SLB platform can preserve both high membrane fluidity and high accessibility to the outside environment, which have never been simultaneously achieved before. Incorporating the built platforms with some surface analytical tools could open the bottleneck of building highly robust in vitro cell-membrane-related bioassays.

Mechanism of unassisted ion transport across membrane bilayers

NASA Technical Reports Server (NTRS)

Wilson, M. A.; Pohorille, A.

1996-01-01

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

Single-particle fusion of influenza viruses reveals complex interactions with target membranes

NASA Astrophysics Data System (ADS)

van der Borg, Guus; Braddock, Scarlett; Blijleven, Jelle S.; van Oijen, Antoine M.; Roos, Wouter H.

2018-05-01

The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.

An Investigation on bilayer structures of electrospun polyacrylonitrile nanofibrous membrane and cellulose membrane used as filtration media for apple juice clarification

NASA Astrophysics Data System (ADS)

Sawitri, Asti; Miftahul Munir, Muhammad; Edikresnha, Dhewa; Sandi, Ahzab; Fauzi, Ahmad; Rajak, Abdul; Natalia, Dessy; Khairurrijal, Khairurrijal

2018-05-01

Nanofibrous membrane has a potential to use in filtration technology with electrospinning as one of the techniques used in synthesizing nanofibers. Polyacrylonitrile (PAN) nanofibrous membranes with various fibers diameters were electrospun by varying its precursor solution concentration. The average fibers diameters of the PAN nanofibrous membranes obtained from the precursor solution concentrations of 6, 9, 12, and 14 wt% were 341, 534, 1274, and 2107 nm, respectively. Filtration media for apple juice clarification were bilayer-structured membranes made of PAN nanofibrous membranes on commercial cellulose microfibrous membranes. It has been shown that the reduction of apple juice color or turbidity performed by the cellulose microfibrous membrane was well enhanced by the presence of the PAN nanofibrous membrane in the bilayer-structured membrane. In addition, the apple-juice color and turbidity reductions increased with decreasing the average fibers diameter of the PAN nanofibrous membrane. Furthermore, the PAN nanofibrous membrane also helped the cellulose microfibrous membrane in the bilayer-structured membrane enhance the reductions of total phenols, protein, and glucose of the apple juice.

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

PubMed Central

MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

2015-01-01

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

Helix formation and stability in membranes.

PubMed

McKay, Matthew J; Afrose, Fahmida; Koeppe, Roger E; Greathouse, Denise V

2018-02-13

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides. Nevertheless, the spontaneous folding process is not well understood at the molecular level. Recent findings suggest that helix fraying and loop formation may be important for overall structure, dynamics and regulation of function. Several types of membrane helices with ionizable amino acids change their topology with pH. Additionally we note that some peptides, including many that are rich in arginine, and a particular analogue of gramicidin, are able passively to translocate across cell membranes. The findings indicate that a final protein structure in a lipid-bilayer membrane is sequence-based, with lipids contributing to stability and regulation. While much progress has been made toward understanding the folding process for alpha-helical membrane proteins, it remains a work in progress. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018 Elsevier B.V. All rights reserved.

Lipid self-assembly and lectin-induced reorganization of the plasma membrane.

PubMed

Sych, Taras; Mély, Yves; Römer, Winfried

2018-05-26

The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears-globally speaking-homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Interactions between antimicrobial polynorbornenes and phospholipid vesicles monitored by light scattering and microcalorimetry.

PubMed

Gabriel, Gregory J; Pool, Joanna G; Som, Abhigyan; Dabkowski, Jeffrey M; Coughlin, E Bryan; Muthukumar, M; Tew, Gregory N

2008-11-04

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S>0, Delta H>0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.

Probing the Interaction between Nanoparticles and Lipid Membranes by Quartz Crystal Microbalance with Dissipation Monitoring

PubMed Central

Yousefi, Nariman; Tufenkji, Nathalie

2016-01-01

There is increasing interest in using quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the interaction of nanoparticles (NPs) with model surfaces. The high sensitivity, ease of use and the ability to monitor interactions in real-time has made it a popular technique for colloid chemists, biologists, bioengineers, and biophysicists. QCM-D has been recently used to probe the interaction of NPs with supported lipid bilayers (SLBs) as model cell membranes. The interaction of NPs with SLBs is highly influenced by the quality of the lipid bilayers. Unlike many surface sensitive techniques, by using QCM-D, the quality of SLBs can be assessed in real-time, hence QCM-D studies on SLB-NP interactions are less prone to the artifacts arising from bilayers that are not well formed. The ease of use and commercial availability of a wide range of sensor surfaces also have made QCM-D a versatile tool for studying NP interactions with lipid bilayers. In this review, we summarize the state-of-the-art on QCM-D based techniques for probing the interactions of NPs with lipid bilayers. PMID:27995125

The three-dimensional structure of aquaporin-1

NASA Astrophysics Data System (ADS)

Walz, Thomas; Hirai, Teruhisa; Murata, Kazuyoshi; Heymann, J. Bernard; Mitsuoka, Kaoru; Fujiyoshi, Yoshinori; Smith, Barbara L.; Agre, Peter; Engel, Andreas

1997-06-01

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6Å resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning α-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

Biomimetic membrane arrays on cast hydrogel supports.

PubMed

Roerdink Lander, Monique; Ibragimova, Sania; Rein, Christian; Vogel, Jörg; Stibius, Karin; Geschke, Oliver; Perry, Mark; Hélix-Nielsen, Claus

2011-06-07

Lipid bilayers are intrinsically fragile and require mechanical support in technical applications based on biomimetic membranes. Tethering the lipid bilayer membranes to solid substrates, either directly through covalent or ionic substrate-lipid links or indirectly on substrate-supported cushions, provides mechanical support but at the cost of small molecule transport through the membrane-support sandwich. To stabilize biomimetic membranes while allowing transport through a membrane-support sandwich, we have investigated the feasibility of using an ethylene tetrafluoroethylene (ETFE)/hydrogel sandwich as the support. The sandwich is realized as a perforated surface-treated ETFE film onto which a hydrogel composite support structure is cast. We report a simple method to prepare arrays of lipid bilayer membranes with low intrinsic electrical conductance on the highly permeable, self-supporting ETFE/hydrogel sandwiches. We demonstrate how the ETFE/hydrogel sandwich support promotes rapid self-thinning of lipid bilayers suitable for hosting membrane-spanning proteins.

Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

PubMed Central

Anong, William A.; Franco, Taina; Chu, Haiyan; Weis, Tahlia L.; Devlin, Emily E.; Bodine, David M.; An, Xiuli; Mohandas, Narla

2009-01-01

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane. PMID:19567882

Membrane architectures for ion-channel switch-based electrochemical biosensors

DOEpatents

Sansinena, Jose-Maria; Redondo, Antonio; Swanson, Basil I.; Yee, Chanel Kitmon; Sapuri/Butti, Annapoorna R.; Parikh, Atul N.; Yang, Calvin

2008-10-28

The present invention is directed to a process of forming a bilayer lipid membrane structure by depositing an organic layer having a defined surface area onto an electrically conductive substrate, removing portions of said organic layer upon said electrically conductive substrate whereby selected portions of said organic layer are removed to form defined voids within said defined surface area of said organic layer and defined islands of organic layer upon said electrically conductive substrate, and, depositing a bilayer lipid membrane over the defined voids and defined islands of organic layer upon said substrate whereby aqueous reservoirs are formed between said electrically conductive substrate and said bilayer lipid membrane, said bilayer lipid membrane characterized as spanning across the defined voids between said defined islands. A lipid membrane structure is also described together with an array of such lipid membrane structure.

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.

Structure and electrical properties of DNA nanotubes embedded in lipid bilayer membranes

PubMed Central

Maiti, Prabal K

2018-01-01

Abstract Engineering the synthetic nanopores through lipid bilayer membrane to access the interior of a cell is a long persisting challenge in biotechnology. Here, we demonstrate the stability and dynamics of a tile-based 6-helix DNA nanotube (DNT) embedded in POPC lipid bilayer using the analysis of 0.2 μs long equilibrium MD simulation trajectories. We observe that the head groups of the lipid molecules close to the lumen cooperatively tilt towards the hydrophilic sugar-phosphate backbone of DNA and form a toroidal structure around the patch of DNT protruding in the membrane. Further, we explore the effect of ionic concentrations to the in-solution structure and stability of the lipid-DNT complex. Transmembrane ionic current measurements for the constant electric field MD simulation provide the I-V characteristics of the water filled DNT lumen in lipid membrane. With increasing salt concentrations, the measured values of transmembrane ionic conductance of the porous DNT lumen vary from 4.3 to 20.6 nS. Simulations of the DNTs with ssDNA and dsDNA overhangs at the mouth of the pore show gating effect with remarkable difference in the transmembrane ionic conductivities for open and close state nanopores. PMID:29136243

Effects of cholesterol depletion on membrane nanostructure in MCF-7 cells by atomic force microscopy

NASA Astrophysics Data System (ADS)

Wang, Yuhua; Jiang, Ningcheng; Shi, Aisi; Zheng, Liqin; Yang, Hongqin; Xie, Shusen

2017-02-01

The cell membrane is composed of phospholipids, glycolipids, cholesterol and proteins that are dynamic and heterogeneous distributed in the bilayer structure and many researches have showed that the plasma membrane in eukaryotic cells contains microdomains termed "lipid raft" in which cholesterol, sphingolipids and specific membrane proteins are enriched. Cholesterol extraction induced lipid raft disruption is one of the most widely used methods for lipid raft research and MβCD is a type of solvent to extract the cholesterol from cell membranes. In this study, the effect of MβCD treatment on the membrane nanostructure in MCF-7 living cells was investigated by atomic force microscopy. Different concentrations of MβCD were selected to deplete cholesterol for 30 min and the viability of cells was tested by MTT assay to obtain the optimal concentration. Then the nanostructure of the cell membrane was detected. The results show that an appropriate concentration of MβCD can induce the alteration of cell membranes nanostructure and the roughness of membrane surface decreases significantly. This may indicate that microdomains of the cell membrane disappear and the cell membrane appears more smoothly. Cholesterol can affect nanostructure and inhomogeneity of the plasma membrane in living cells.

Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1)

PubMed Central

2017-01-01

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400–Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 μs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a “pulling in” of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments. PMID:28068080

Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1).

PubMed

Fowler, Philip W; Sansom, Mark S P; Reithmeier, Reinhart A F

2017-02-07

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400-Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 μs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a "pulling in" of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments.

Effect of the interfacial tension and ionic strength on the thermodynamic barrier associated to the benzocaine insertion into a cell membrane.

PubMed

López Cascales, J J; Oliveira Costa, S D

2013-02-01

The insertion of local anaesthetics into a cell membrane is a key aspect for explaining their activity at a molecular level. It has been described how the potency and response time of local anaesthetics is improved (for clinical applications) when they are dissolved in a solution of sodium bicarbonate. With the aim of gaining insight into the physico-chemical principles that govern the action mechanism of these drugs at a molecular level, simulations of benzocaine in binary lipid bilayers formed by DPPC/DPPS were carried out for different ionic strengths of the aqueous solution. From these molecular dynamic simulations, we observed how the thermodynamic barrier associated with benzocaine insertion into the lipid bilayers diminished exponentially as the fraction of DPPS in the bilayer increased, especially when the ionic strength of the aqueous solution increased. In line with these results, we also observed how this thermodynamic barrier diminished exponentially with the phospholipid/water interfacial tension. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of benzocaine and propranolol on phospholipid-based bilayers.

PubMed

Mangiapia, G; Gvaramia, M; Kuhrts, L; Teixeira, J; Koutsioubas, A; Soltwedel, O; Frielinghaus, H

2017-12-06

Cell membranes play a fundamental role in protecting the cell from its surroundings, in addition to hosting many proteins with fundamental biological tasks. A study of drug/lipid interactions is a necessary and important step in fully clarifying the role and action mechanism of active ingredients, and shedding light on possible complications caused by drug overdosage. In this paper, the influence of benzocaine and propranolol drugs on the structure of l-α-phosphatidylcholine-based membranes has been investigated by means of neutron reflectivity, grazing incidence small angle neutron scattering, and small/ultra-small angle neutron scattering. Investigations allowed discovering a stiffening of the membranes and the formation of stalks, caused by the presence of benzocaine. On the other hand, disordered bilayers (lamellar powders) and highly curved structures were found in the presence of propranolol. The results obtained may be rationalized in terms of the molecular structures of drugs and may serve as a starting point for explaining the toxic behavior in long-term and overdosage scenarios.

How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

NASA Astrophysics Data System (ADS)

Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Fang, Weihai

2012-02-01

In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

NASA Astrophysics Data System (ADS)

Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

2013-12-01

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

PLGA/liposome hybrid nanoparticles for short-chain ceramide delivery.

PubMed

Zou, Peng; Stern, Stephan T; Sun, Duxin

2014-03-01

Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly (lactic-coglycolicacid) (PLGA). BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor). FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 min. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 h. The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs.

A stable planar bilayer membrane of phospholipid supported by cellulose sheets.

PubMed

Setaka, M; Yamamoto, T; Sato, N; Yano, M; Kwan, T

1982-01-01

A new method is reported for preparing a thin planar membrane of 1,2-distearoylsn-glycero-3-phosphocholine and egg yolk lecithin-cholesterol (molar ratio of 1:1) between a pair of cellulose sheets. This technique, developed from the method of the multilayer planar membrane preparation (Setaka, M., et al. (1979) J. Biochem. 86, 355-362; 1619-1622; (1980) J. Biochem. 88, 1819-1829), consisted of three experimental processes. First, a phospholipid monolayer was prepared at an air-water interface, then taken up on a stretched cellulose sheet. A thin lipid membrane, supported from both sides by cellulose sheets, was constructed by combining two of these lipid monolayer-cellulose sheets. The permeability coefficient of the thin lipid membrane was estimated by removing the effect of two outer cellulose sheets, and this permeability was found to be larger than those of other model membranes of a lipid bilayer, indicating that the present lipid membrane is not a perfect single lipid bilayer. However, certain experimental evidence suggests that the bulk of the phospholipids formed a bilayer between the two cellulose sheets. Since this lipid membrane is particularily stable, larger membranes can be prepared by the present method than other planar bilayer membranes of lipid, which are usually constructed inside a pin hole in a thin teflon sheet.

Arrayed water-in-oil droplet bilayers for membrane transport analysis.

PubMed

Watanabe, R; Soga, N; Hara, M; Noji, H

2016-08-02

The water-in-oil droplet bilayer is a simple and useful lipid bilayer system for membrane transport analysis. The droplet interface bilayer is readily formed by the contact of two water-in-oil droplets enwrapped by a phospholipid monolayer. However, the size of individual droplets with femtoliter volumes in a high-throughput manner is difficult to control, resulting in low sensitivity and throughput of membrane transport analysis. To overcome this drawback, in this study, we developed a novel micro-device in which a large number of droplet interface bilayers (>500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate. The droplet volume was controllable from 3.5 to 350 fL by changing the hydrophobic/hydrophilic pattern on the device, allowing high-throughput analysis of membrane transport mechanisms including membrane permeability to solutes (e.g., ions or small molecules) with or without the aid of transport proteins. Thus, this novel platform broadens the versatility of water-in-oil droplet bilayers and will pave the way for novel analytical and pharmacological applications such as drug screening.

Modeling Nanoparticle Wrapping or Translocation in Bilayer Membranes

PubMed Central

Curtis, Emily M.; Bahrami, Amir H.; Weikl, Thomas R.; Hall, Carol K.

2015-01-01

The spontaneous wrapping of nanoparticles by membranes is of increasing interest as nanoparticles become more prevalent in consumer products and hence more likely to enter the human body. We introduce a simulations-based tool that can be used to visualize the molecular level interaction between nanoparticles and bilayer membranes. By combining LIME, an intermediate resolution, implicit solvent model for phospholipids, with discontinuous molecular dynamics (DMD), we are able to simulate the wrapping or embedding of nanoparticles by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer membranes. Simulations of hydrophilic nanoparticles with diameters from 10Å to 250Å show that hydrophilic nanoparticles with diameters greater than 20Å become wrapped while the nanoparticle with a diameter of 10Å does not . Instead this smaller particle became embedded in the bilayer surface where it could interact with the hydrophilic head groups of the lipid molecules. We also investigate the interaction between a DPPC bilayer and hydrophobic nanoparticles with diameters 10Å to 40Å. These nanoparticles do not undergo the wrapping process; instead they directly penetrate the membrane and embed themselves within the inner hydrophobic core of the bilayers. PMID:26260123

Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers

NASA Astrophysics Data System (ADS)

Lewis, Shanta

Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4microg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4microg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.

The Integrin Receptor in Biologically Relevant Bilayers: Insights from Molecular Dynamics Simulations.

PubMed

Kalli, Antreas C; Rog, Tomasz; Vattulainen, Ilpo; Campbell, Iain D; Sansom, Mark S P

2017-08-01

Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2-F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2-F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes.

Detection of superlattice domain formation in ternary lipid mixtures using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Mutlu, Burcin; Lopez, Stephanie; Vaughn, Mark; Huang, Juyang; Cheng, K.

2011-10-01

Multicomponent lipid bilayers represent an important model system for studying the structures and functions of cell membranes. At present, the lateral organization of lipid components, particularly the formation of regular distribution, in lipid membranes containing charged lipid, e.g., phosphatidylserine, is not clear. Using a ternary phosphatidylcholine/phosphatidylserine/cholesterol lipid bilayer system, the presence of ordered domain formation was examined by measuring the fluorescence anisotropy of the embedded fluorescent probe, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)amino)-23,24-bisnor-5-cholen-3β- ol (NBD-CHOL), with structure similar to that of a cholesterol, as a function of phospatidylserine composition. The plot of the anisotropy vs. phosphatidylserine revealed abrupt changes at certain critical compositions of phosphatidylserine. Some of these critical compositions agree favorably with those predicted by the headgroup superlattice model suggesting that the charged phosphatidylserine lipid molecules adopt a superlattice-like distribution in the lipid bilayer at some predicted compositions. The ordered distribution of charged lipids may play an important role in the regulation of the composition of the biological membranes.

CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin

PubMed Central

van Pee, Katharina; Neuhaus, Alexander; D'Imprima, Edoardo; Mills, Deryck J; Kühlbrandt, Werner; Yildiz, Özkan

2017-01-01

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane. DOI: http://dx.doi.org/10.7554/eLife.23644.001 PMID:28323617

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

PubMed Central

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S.; Stenvang, Marcel; Otzen, Daniel E.

2014-01-01

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. PMID:25304506

The antimicrobial mechanism of action of epsilon-poly-l-lysine.

PubMed

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S; Stenvang, Marcel; Otzen, Daniel E; Meyer, Rikke L

2014-12-01

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Placing and shaping liposomes with reconfigurable DNA nanocages

NASA Astrophysics Data System (ADS)

Zhang, Zhao; Yang, Yang; Pincet, Frederic; C. Llaguno, Marc; Lin, Chenxiang

2017-07-01

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

Placing and shaping liposomes with reconfigurable DNA nanocages.

PubMed

Zhang, Zhao; Yang, Yang; Pincet, Frederic; Llaguno, Marc C; Lin, Chenxiang

2017-06-23

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

Visualization of Detergent Solubilization of Membranes: Implications for the Isolation of Rafts

PubMed Central

Garner, Ashley E.; Smith, D. Alastair; Hooper, Nigel M.

2008-01-01

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (ld) and raft liquid ordered (lo) lipid phases by selectively solubilizing the ld phase. A higher concentration of Lubrol was required, and not all the ld phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some ld phase and then progressed to the solubilization of both ld and lo phases simultaneously. Octyl glucoside simultaneously solubilized both lo and ld phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems. PMID:17933878

Visualization of detergent solubilization of membranes: implications for the isolation of rafts.

PubMed

Garner, Ashley E; Smith, D Alastair; Hooper, Nigel M

2008-02-15

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.

How sterol tilt regulates properties and organization of lipid membranes and membrane insertions

PubMed Central

Khelashvili, George; Harries, Daniel

2013-01-01

Serving as a crucial component of mammalian cells, cholesterol critically regulates the functions of biomembranes. This review focuses on a specific property of cholesterol and other sterols: the tilt modulus χ that quantifies the energetic cost of tilting sterol molecules inside the lipid membrane. We show how χ is involved in determining properties of cholesterol-containing membranes, and detail a novel approach to quantify its value from atomistic molecular dynamics (MD) simulations. Specifically, we link χ with other structural, thermodynamic, and mechanical properties of cholesterol-containing lipid membranes, and delineate how this useful parameter can be obtained from the sterol tilt probability distributions derived from relatively small-scale unbiased MD simulations. We demonstrate how the tilt modulus quantitatively describes the aligning field that sterol molecules create inside the phospholipid bilayers, and we relate χ to the bending rigidity of the lipid bilayer through effective tilt and splay energy contributions to the elastic deformations. Moreover, we show how χ can conveniently characterize the “condensing effect” of cholesterol on phospholipids. Finally, we demonstrate the importance of this cholesterol aligning field to the proper folding and interactions of membrane peptides. Given the relative ease of obtaining the tilt modulus from atomistic simulations, we propose that χ can be routinely used to characterize the mechanical properties of sterol/lipid bilayers, and can also serve as a required fitting parameter in multi-scaled simulations of lipid membrane models to relate the different levels of coarse-grained details. PMID:23291283

Molecular modeling of biomembranes and their complexes with protein transmembrane α-helices

NASA Astrophysics Data System (ADS)

Kuznetsov, Andrey S.; Smirnov, Kirill V.; Antonov, Mikhail Yu.; Nikolaev, Ivan N.; Efremov, Roman G.

2017-11-01

Helical segments are common structural elements of membrane proteins. Dimerization and oligomerization of transmembrane (TM) α-helices provides the framework for spatial structure formation and protein-protein interactions. The membrane itself also takes part in the protein functioning. There are some examples of the mutual influence of the lipid bilayer properties and embedded membrane proteins. This work aims at the detail investigation of protein-lipid interactions using model systems: TM peptides corresponding to native protein segments. Three peptides were considered corresponding to TM domains of human glycophorin A (GpA), epidermal growth factor receptor (EGFR) and proposed TM-segment of human neuraminidase-1 (Neu1). A computational analysis of structural and dynamical properties was performed using molecular dynamics method. Monomers of peptides were considered incorporated into hydrated lipid bilayers. It was confirmed, that all these TM peptides have stable helical conformation in lipid environment, and the mutual adaptation of peptides and membrane was observed. It was shown that incorporation of the peptide into membrane results in the modulation of local and mean structural properties of the bilayer. Each peptide interacts with lipid acyl chains having special binding sites on the surface of central part of α-helix that exist for at least 200 ns. However, lipid acyl chains substitute each other faster occupying the same site. The formation of a special pattern of protein-lipid interactions may modulate the association of TM domains of membrane proteins, so membrane environment should be considered when proposing new substances targeting cell receptors.

Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface

PubMed Central

Cheng, Sara Y.; Chou, George; Buie, Creighton; Vaughn, Mark W.; Compton, Campbell; Cheng, Kwan H.

2016-01-01

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein–lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein–protein but weaker protein–lipid interactions for a dimeric protein on membrane surfaces. PMID:26827904

Dendrimer Interactions with Lipid Bilayer: Comparison of Force Field and Effect of Implicit vs Explicit Solvation.

PubMed

Kanchi, Subbarao; Gosika, Mounika; Ayappa, K G; Maiti, Prabal K

2018-06-13

The understanding of dendrimer interactions with cell membranes has great importance in drug/gene delivery based therapeutics. Although molecular simulations have been used to understand the nature of dendrimer interactions with lipid membranes, its dependency on available force field parameters is poorly understood. In this study, we have carried out fully atomistic molecular dynamics (MD) simulations of a protonated G3 poly(amido amine) (PAMAM) dendrimer-dimyristoylphosphatidylcholine (DMPC) lipid bilayer complex using three different force fields (FFs) namely, CHARMM, GAFF, and GROMOS in the presence of explicit water to understand the structure of the lipid-dendrimer complex and nature of their interaction. CHARMM and GAFF dendrimers initially in contact with the lipid head groups were found to move away from the lipid bilayer during the course of simulation; however, the dendrimer remained strongly bound to the lipid head groups with the GROMOS FF. Potential of the mean force (PMF) computations of the dendrimer along the bilayer normal showed a repulsive barrier (∼20 kcal/mol) between dendrimer and lipid bilayer in the case of CHARMM and GAFF force fields. In contrast, an attractive interaction (∼40 kcal/mol) is obtained with the GROMOS force field, consistent with experimental observations of membrane binding observed with lower generation G3 PAMAM dendrimers. This difference with the GROMOS dendrimer is attributed to the strong dendrimer-lipid interaction and lowered surface hydration of the dendrimer. Assessing the role of solvent, we find that the CHARMM and GAFF dendrimers strongly bind to the lipid bilayer with an implicit solvent (Generalized Born) model, whereas binding is not observed with explicit water (TIP3P). The opposing nature of dendrimer-membrane interactions in the presence of explicit and implicit solvents demonstrates that hydration effects play an important role in modulating the dendrimer-lipid interaction warranting a case for refinement of the existing dendrimer/lipid force fields.

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Mechanics of Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Powers, Thomas R.

All cells have membranes. The plasma membrane encapsulates the cell's interior, acting as a barrier against the outside world. In cells with nuclei (eukaryotic cells), membranes also form internal compartments (organelles) which carry out specialized tasks, such as protein modification and sorting in the case of the Golgi apparatus, and ATP production in the case of mitochondria. The main components of membranes are lipids and proteins. The proteins can be channels, carriers, receptors, catalysts, signaling molecules, or structural elements, and typically contribute a substantial fraction of the total membrane dry weight. The equilibrium properties of pure lipid membranes are relatively well-understood, and will be the main focus of this article. The framework of elasticity theory and statistical mechanics that we will develop will serve as the foundation for understanding biological phenomena such as the nonequilibrium behavior of membranes laden with ion pumps, the role of membrane elasticity in ion channel gating, and the dynamics of vesicle fission and fusion. Understanding the mechanics of lipid membranes is also important for drug encapsulation and delivery.

Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

PubMed

Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman

2017-01-01

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Hydrodynamic coupling of particle inclusions embedded in curved lipid bilayer membranes

DOE PAGES

Sigurdsson, Jon Karl; Atzberger, Paul J.

2016-06-27

Here, we develop theory and computational methods to investigate particle inclusions embedded within curved lipid bilayer membranes. We consider the case of spherical lipid vesicles where inclusion particles are coupled through (i) intramembrane hydrodynamics, (ii) traction stresses with the external and trapped solvent fluid, and (iii) intermonolayer slip between the two leaflets of the bilayer. We investigate relative to flat membranes how the membrane curvature and topology augment hydrodynamic responses. We show how both the translational and rotational mobility of protein inclusions are effected by the membrane curvature, ratio of intramembrane viscosity to solvent viscosity, and intermonolayer slip. For generalmore » investigations of many-particle dynamics, we also discuss how our approaches can be used to treat the collective diffusion and hydrodynamic coupling within spherical bilayers.« less

Hydrodynamic coupling of particle inclusions embedded in curved lipid bilayer membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigurdsson, Jon Karl; Atzberger, Paul J.

Here, we develop theory and computational methods to investigate particle inclusions embedded within curved lipid bilayer membranes. We consider the case of spherical lipid vesicles where inclusion particles are coupled through (i) intramembrane hydrodynamics, (ii) traction stresses with the external and trapped solvent fluid, and (iii) intermonolayer slip between the two leaflets of the bilayer. We investigate relative to flat membranes how the membrane curvature and topology augment hydrodynamic responses. We show how both the translational and rotational mobility of protein inclusions are effected by the membrane curvature, ratio of intramembrane viscosity to solvent viscosity, and intermonolayer slip. For generalmore » investigations of many-particle dynamics, we also discuss how our approaches can be used to treat the collective diffusion and hydrodynamic coupling within spherical bilayers.« less

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

PubMed

Elías-Wolff, Federico; Lindén, Martin; Lyubartsev, Alexander P; Brandt, Erik G

2018-03-13

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

PubMed Central

Discher, D E; Mohandas, N

1996-01-01

Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:8889146

Effects of carotenoids on lipid bilayers.

PubMed

Johnson, Quentin R; Mostofian, Barmak; Fuente Gomez, Gabriel; Smith, Jeremy C; Cheng, Xiaolin

2018-01-31

Carotenoids have been found to be important in improving the integrity of biomembranes in eukaryotes. However, the molecular details of how carotenoids modulate the physical properties of biomembranes are unknown. To this end, we have conducted a series of molecular dynamics simulations of different biologically-relevant membranes in the presence of carotenoids. The carotenoid effect on the membrane was found to be specific to the identity of the carotenoid and the composition of the membrane itself. Therefore, different classes of carotenoids produce a different effect on the membrane, and different membrane phases are affected differently by carotenoids. It is apparent from our data that carotenoids do trigger the bilayer to become thinner. The mechanism by which this occurs depends on two competing factors, the ability of the lipid tails of opposing monolayers to either (1) compress or (2) interdigitate as the bilayer condenses. Indeed, carotenoids directly influence the physical properties via these two mechanisms, thus compacting the bilayer. However, the degree to which these competing mechanisms are utilized depends on the bilayer phase and the carotenoid identity.

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

PubMed

MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

2015-04-24

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Engineering monolayer poration for rapid exfoliation of microbial membranes.

PubMed

Pyne, Alice; Pfeil, Marc-Philipp; Bennett, Isabel; Ravi, Jascindra; Iavicoli, Patrizia; Lamarre, Baptiste; Roethke, Anita; Ray, Santanu; Jiang, Haibo; Bella, Angelo; Reisinger, Bernd; Yin, Daniel; Little, Benjamin; Muñoz-García, Juan C; Cerasoli, Eleonora; Judge, Peter J; Faruqui, Nilofar; Calzolai, Luigi; Henrion, Andre; Martyna, Glenn J; Grovenor, Chris R M; Crain, Jason; Hoogenboom, Bart W; Watts, Anthony; Ryadnov, Maxim G

2017-02-01

The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that transmembrane poration is not necessary for antimicrobial activity and reveal a distinct poration mechanism that targets the outer leaflet of phospholipid bilayers. Using a combination of molecular-scale and real-time imaging, spectroscopy and spectrometry approaches, we introduce a structural motif with a universal insertion mode in reconstituted membranes and live bacteria. We demonstrate that this motif rapidly assembles into monolayer pits that coalesce during progressive membrane exfoliation, leading to bacterial cell death within minutes. The findings offer a new physical basis for designing effective antibiotics.

Surface Plasmon Resonance Study of the Binding of PEO-PPO-PEO Triblock Copolymer and PEO Homopolymer to Supported Lipid Bilayers.

PubMed

Kim, Mihee; Vala, Milan; Ertsgaard, Christopher T; Oh, Sang-Hyun; Lodge, Timothy P; Bates, Frank S; Hackel, Benjamin J

2018-06-12

Poloxamer 188 (P188), a poly(ethylene oxide)- b-poly(propylene oxide)- b-poly(ethylene oxide) triblock copolymer, protects cell membranes against various external stresses, whereas poly(ethylene oxide) (PEO; 8600 g/mol) homopolymer lacks protection efficacy. As part of a comprehensive effort to elucidate the protection mechanism, we used surface plasmon resonance (SPR) to obtain direct evidence of binding of the polymers onto supported lipid bilayers. Binding kinetics and coverage of P188 and PEO were examined and compared. Most notably, PEO exhibited membrane association comparable to that of P188, evidenced by comparable association rate constants and coverage. This result highlights the need for additional mechanistic understanding beyond simple membrane association to explain the differential efficacy of P188 in therapeutic applications.

Rationalization of reduced penetration of drugs through ceramide gel phase membrane.

PubMed

Paloncýová, Markéta; DeVane, Russell H; Murch, Bruce P; Berka, Karel; Otyepka, Michal

2014-11-25

Since computing resources have advanced enough to allow routine molecular simulation studies of drug molecules interacting with biologically relevant membranes, a considerable amount of work has been carried out with fluid phospholipid systems. However, there is very little work in the literature on drug interactions with gel phase lipids. This poses a significant limitation for understanding permeation through the stratum corneum where the primary pathway is expected to be through a highly ordered lipid matrix. To address this point, we analyzed the interactions of p-aminobenzoic acid (PABA) and its ethyl (benzocaine) and butyl (butamben) esters with two membrane bilayers, which differ in their fluidity at ambient conditions. We considered a dioleoylphosphatidylcholine (DOPC) bilayer in a fluid state and a ceramide 2 (CER2, ceramide NS) bilayer in a gel phase. We carried out unbiased (100 ns long) and biased z-constraint molecular dynamics simulations and calculated the free energy profiles of all molecules along the bilayer normal. The free energy profiles converged significantly slower for the gel phase. While the compounds have comparable affinities for both membranes, they exhibit penetration barriers almost 3 times higher in the gel phase CER2 bilayer. This elevated barrier and slower diffusion in the CER2 bilayer, which are caused by the high ordering of CER2 lipid chains, explain the low permeability of the gel phase membranes. We also compared the free energy profiles from MD simulations with those obtained from COSMOmic. This method provided the same trends in behavior for the guest molecules in both bilayers; however, the penetration barriers calculated by COSMOmic did not differ between membranes. In conclusion, we show how membrane fluid properties affect the interaction of drug-like molecules with membranes.

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes.

PubMed

Rakers, Lena; Grill, David; Matos, Anna L L; Wulff, Stephanie; Wang, Da; Börgel, Jonas; Körsgen, Martin; Arlinghaus, Heinrich F; Galla, Hans-Joachim; Gerke, Volker; Glorius, Frank

2018-05-01

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Changes in membrane lipid composition during saline growth of the fresh water cyanobacterium Synechococcus 6311

NASA Technical Reports Server (NTRS)

Huflejt, M. E.; Tremolieres, A.; Pineau, B.; Lang, J. K.; Hatheway, J.; Packer, L.

1990-01-01

Growth of Synechococcus 6311 in the presence of 0.5 molar NaCl is accompanied by significant changes in membrane lipid composition. Upon transfer of the cells from a low salt' (0.015 molar NaCl) to high salt' (0.5 molar NaCl) growth medium at different stages of growth, a rapid decrease in palmitoleic acid (C16:1 delta 9) content was accompanied by a concomitant increase in the amount of the two C18:1 acids (C18:1 delta 9, C18:1 delta 11), with the higher increase in oleic acid C18:1 delta 9 content. These changes began to occur within the first hour after the sudden elevation of NaCl and progressed for about 72 hours. The percentage of palmitic acid (C16:0) and stearic acid (C18:0) remained almost unchanged in the same conditions. High salt-dependent changes within ratios of polar lipid classes also occurred within the first 72 hours of growth. The amount of monogalactosyl diacylglycerol (bilayer-destabilizing lipid) decreased and that of the digalactosyl diacylglycerol (bilayer-stabilizing lipid) increased. Consequently, in the three day old cells, the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol in the membranes of high salt-grown cells was about half of that in the membranes of low salt-grown cells. The total content of anionic lipids (phosphatidylglycerol and sulfoquinovosyl diacylglycerol) was always higher in the isolated membranes and the whole cells from high salt-grown cultures compared to that in the cells and membranes from low salt-grown cultures. All the observed rearrangements in the lipid environment occurred in both thylakoid and cytoplasmic membranes. Similar lipid composition changes, however, to a much lesser extent, were also observed in the aging, low salt-grown cultures. The observed changes in membrane fatty acids and lipids composition correlate with the alterations in electron and ion transport activities, and it is concluded that the rearrangement of the membrane lipid environment is an essential part of the process by which cells control membrane function and stability.

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study.

PubMed

Karami, Leila; Jalili, Seifollah

2015-01-01

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug-bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25 ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug-bilayer interaction is crucial for the liposomal drug design.

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.

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

PubMed

Odinokov, Alexey; Ostroumov, Denis

2015-12-03

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.

Membrane Protein Incorporation into Nano-Bioelectronics: An insight into Rhodopsin Controlled SiNW-FET Devices

NASA Astrophysics Data System (ADS)

Tunuguntla, Ramya

Biological systems use different energy sources to interact with their environments by creating ion gradients, membrane electric potentials, or a proton motive force to accomplish strikingly complex tasks on the nanometer length scale, such as energy harvesting, and whole organism replication. Most of this activity involves a vast arsenal of active and passive ion channels, membrane receptors and ion pumps that mediate complex and precise transport across biological membranes. Despite the remarkable rate of progress exhibited by modern microelectronic devices, they still cannot compete with the efficiency and precision of biological systems on the component level. At the same time, the sophistication of these molecular machines provides an excellent opportunity to use them in hybrid bioelectronic devices where such a combination could deliver enhanced electronic functionality and enable seamless bi-directional interfaces between man-made and biological assemblies. Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex-vivo devices such as electronic biosensors, thin-film protein arrays, or bio-fuel cells. Since most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In our work, we have explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes. One-dimensional inorganic nanostructures, which have critical dimensions comparable to the sizes of biological molecules, form an excellent materials platform for building such integrated structures. Researchers already use silicon nanowire-based field effect transistors functionalized with molecular recognition sites in a diverse array of biosensors. In our group, we have been developing a platform for integration of membrane protein functionality and electronic devices using a 1-D phospholipid bilayer device architecture. In these devices, the membrane proteins reside within the lipid bilayer that covers a nanowire channel of a field-effect transistor. This lipid bilayer performs several functions: it shields the nanowire from the solution species; it serves as a native-like environment for membrane proteins and preserves their functionality, integrity, and even vectorality. In this work, we show that a 1-D bilayer device incorporating a rhodopsin proton pump allows us to couple light-driven proton transport to a bioelectronic circuit. We also report that we were able to adapt another distinctive feature of biological signal processing---their widespread use of modifiers, co-factors, and mediator molecules---to regulate and fine-tune the operational characteristics of the bioelectronic device. In our example, we use co-assembly of protein channels and ionophores in the 1-D bilayer to modify the device output levels and response time.

Transmembrane protein diffusion in gel-supported dual-leaflet membranes.

PubMed

Wang, Chih-Ying; Hill, Reghan J

2014-11-18

Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed permeability and solvent viscosity. For asymmetric configurations, i.e., supports with contrasting permeability, as realized for cells in contact with hydrogel scaffolds or culture media, the diffusion coefficient can reflect interleaflet friction. Reasonable approximations, for sufficiently large tracers on low-permeability supports, are furnished by a recent phenomenological theory from the literature. Interpreting literature data, albeit for hard-supported membranes, provides a theoretical basis for the phenomenological Stokes drag law as well as strengthening assertions that nonhydrodynamic interactions are important in supported bilayer systems, possibly leading to overestimates of the membrane/leaflet viscosity. Our theory provides a theoretical foundation for future experimental studies of tracer diffusion in gel-supported membranes.

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.

Synthetic ion channels via self-assembly: a route for embedding porous polyoxometalate nanocapsules in lipid bilayer membranes.

PubMed

Carr, Rogan; Weinstock, Ira A; Sivaprasadarao, Asipu; Müller, Achim; Aksimentiev, Aleksei

2008-11-01

Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels; however, their use as an artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this Letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na(+) cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane.

Synthetic Ion Channels via Self-Assembly: a Route for Embedding Porous Polyoxometalate Nanocapsules in Lipid Bilayer Membranes

PubMed Central

Carr, Rogan; Weinstock, Ira A.; Sivaprasadarao, Asipu; Müller, Achim; Aksimentiev, Aleksei

2010-01-01

Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels, however, their use as artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na+ cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane. PMID:18844424

Thermodynamic free energy methods to investigate shape transitions in bilayer membranes.

PubMed

Ramakrishnan, N; Tourdot, Richard W; Radhakrishnan, Ravi

2016-06-01

The conformational free energy landscape of a system is a fundamental thermodynamic quantity of importance particularly in the study of soft matter and biological systems, in which the entropic contributions play a dominant role. While computational methods to delineate the free energy landscape are routinely used to analyze the relative stability of conformational states, to determine phase boundaries, and to compute ligand-receptor binding energies its use in problems involving the cell membrane is limited. Here, we present an overview of four different free energy methods to study morphological transitions in bilayer membranes, induced either by the action of curvature remodeling proteins or due to the application of external forces. Using a triangulated surface as a model for the cell membrane and using the framework of dynamical triangulation Monte Carlo, we have focused on the methods of Widom insertion, thermodynamic integration, Bennett acceptance scheme, and umbrella sampling and weighted histogram analysis. We have demonstrated how these methods can be employed in a variety of problems involving the cell membrane. Specifically, we have shown that the chemical potential, computed using Widom insertion, and the relative free energies, computed using thermodynamic integration and Bennett acceptance method, are excellent measures to study the transition from curvature sensing to curvature inducing behavior of membrane associated proteins. The umbrella sampling and WHAM analysis has been used to study the thermodynamics of tether formation in cell membranes and the quantitative predictions of the computational model are in excellent agreement with experimental measurements. Furthermore, we also present a method based on WHAM and thermodynamic integration to handle problems related to end-point-catastrophe that are common in most free energy methods.

Interaction of injectable neurotropic drugs with the red cell membrane.

PubMed

Reinhart, Walter H; Lubszky, Szabina; Thöny, Sandra; Schulzki, Thomas

2014-10-01

The normal red blood cell (RBC) shape is a biconcave discocyte. An intercalation of a drug in the outer half of the membrane lipid bilayer leads to echinocytosis, an intercalation in the inner half to stomatocytosis. We have used the shape transforming capacity of RBCs as a model to analyse the membrane interaction potential of various neurotropic drugs. Chlorpromazine, clomipramine, citalopram, clonazepam, and diazepam induced a reversible stomatocytosis, phenytoin induced echinocytosis, while the anticonvulsants levetiracetam, valproic acid and phenobarbital had no effect. This diversity of RBC shape transformations suggests that the pharmacological action is not linked to the membrane interaction. We conclude that this simple RBC shape transformation assay could be a useful tool to screen for potential drug interactions with cell membranes. Copyright © 2014. Published by Elsevier Ltd.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

PubMed

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

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.

Cholesterol effect on water permeability through DPPC and PSM lipid bilayers: a molecular dynamics study.

PubMed

Saito, Hiroaki; Shinoda, Wataru

2011-12-29

Water permeability of two different lipid bilayers of dipalmitoylphosphatidylcholine (DPPC) and palmitoylsphingomyelin (PSM) in the absence and presence of cholesterol (0-50 mol %) have been studied by molecular dynamics simulations to elucidate the molecular mechanism of the reduction in water leakage across the membranes by the addition of cholesterol. An enhanced free energy barrier was observed in these membranes with increased cholesterol concentration, and this was explained by the reduced cavity density around the cholesterol in the hydrophobic membrane core. There was an increase of trans conformers in the hydrophobic lipid chains adjacent to the cholesterol, which reduced the cavity density. The enhanced free energy barrier was found to be the main reason to reduce the water permeability with increased cholesterol concentration. At low cholesterol concentrations the PSM bilayer exhibited a higher free energy barrier than the DPPC bilayer for water permeation, while at greater than 30 mol % of cholesterol the difference became minor. This tendency for the PSM and DPPC bilayers to resemble each other at higher cholesterol concentrations was similar to commonly observed trends in several structural properties, such as order parameters, cross-sectional area per molecule, and cavity density profiles in the hydrophobic regions of bilayer membranes. These results demonstrate that DPPC and PSM bilayers with high cholesterol contents possess similar physical properties, which suggests that the solubility of cholesterol in these lipid bilayers has importance for an understanding of multicomponent lipid membranes with cholesterol. © 2011 American Chemical Society

Membrane solid-state NMR in Canada: A historical perspective.

PubMed

Auger, Michèle

2017-11-01

This manuscript presents an overview of more than 40years of membrane solid-state nuclear magnetic resonance (NMR) research in Canada. This technique is a method of choice for the study of the structure and dynamics of lipid bilayers; bilayer interactions with a variety of molecules such as membrane peptides, membrane proteins and drugs; and to investigate membrane peptide and protein structure, dynamics, and topology. Canada has a long tradition in this field of research, starting with pioneering work on natural and model membranes in the 1970s in a context of emergence of biophysics in the country. The 1980s and 1990s saw an emphasis on studying lipid structures and dynamics, and peptide-lipid and protein-lipid interactions. The study of bicelles began in the 1990s, and in the 2000s there was a rise in the study of membrane protein structures. Novel perspectives include using dynamic nuclear polarization (DNP) for membrane studies and using NMR in live cells. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Aligning nanodiscs at the air-water interface, a neutron reflectivity study.

PubMed

Wadsäter, Maria; Simonsen, Jens B; Lauridsen, Torsten; Tveten, Erlend Grytli; Naur, Peter; Bjørnholm, Thomas; Wacklin, Hanna; Mortensen, Kell; Arleth, Lise; Feidenhans'l, Robert; Cárdenas, Marité

2011-12-20

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 Å with a surface coverage of 66 ± 4%. This layer is located about 15 Å below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 Å) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer. © 2011 American Chemical Society

Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

PubMed Central

Zattas, Dimitrios; Hochstrasser, Mark

2014-01-01

The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

Bilayered construct for simultaneous regeneration of alveolar bone and periodontal ligament.

PubMed

Nivedhitha Sundaram, M; Sowmya, S; Deepthi, S; Bumgardener, Joel D; Jayakumar, R

2016-05-01

Periodontitis is an inflammatory disease that causes destruction of tooth-supporting tissues and if left untreated leads to tooth loss. Current treatments have shown limited potential for simultaneous regeneration of the tooth-supporting tissues. To recreate the complex architecture of the periodontium, we developed a bilayered construct consisting of poly(caprolactone) (PCL) multiscale electrospun membrane (to mimic and regenerate periodontal ligament, PDL) and a chitosan/2wt % CaSO4 scaffold (to mimic and regenerate alveolar bone). Scanning electron microscopy results showed the porous nature of the scaffold and formation of beadless electrospun multiscale fibers. The fiber diameter of microfiber and nanofibers was in the range of 10 ± 3 µm and 377 ± 3 nm, respectively. The bilayered construct showed better protein adsorption compared to the control. Osteoblastic differentiation of human dental follicle stem cells (hDFCs) on chitosan/2wt % CaSO4 scaffold showed maximum alkaline phosphatase at seventh day followed by a decline thereafter when compared to chitosan control scaffold. Fibroblastic differentiation of hDFCs was confirmed by the expression of PLAP-1 and COL-1 proteins which were more prominent on PCL multiscale membrane in comparison to control membranes. Overall these results show that the developed bilayered construct might serve as a good candidate for the simultaneous regeneration of the alveolar bone and PDL. © 2015 Wiley Periodicals, Inc.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

PubMed

Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas; Chiantia, Salvatore

2017-06-15

Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them. Copyright © 2017 American Society for Microbiology.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

PubMed Central

Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas

2017-01-01

ABSTRACT Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them. PMID:28356535

PLGA/liposome hybrid nanoparticles for short-chain ceramide delivery

PubMed Central

Zou, Peng; Stern, Stephan T.; Sun, Duxin

2014-01-01

Purpose Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly( lactic-coglycolicacid) (PLGA). Methods BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor). Results FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 minutes. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 hours. Conclusions The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs. PMID:24065591

Asymmetric bi-layer PFSA membranes as model systems for the study of water management in the PEMFC.

PubMed

Peng, Z; Peng, A Z; Morin, A; Huguet, P; Lanteri, Y; Deabate, S

2014-10-14

New bi-layer PFSA membranes made of Nafion® NRE212 and Aquivion™ E79-05s with different equivalent weights are prepared with the aim of managing water repartition in the PEMFC. The membrane water transport properties, i.e. back-diffusion and electroosmosis, as well as the electrochemical performances, are compared to those of state-of-art materials. The actual water content (the inner water concentration profile across the membrane thickness) is measured under operation in the fuel cell by in situ Raman microspectroscopy. The orientation of the equivalent weight gradient with respect to the water external gradient and to the proton flow direction affects the membrane water content, the water transport ability and, thus, the fuel cell performances. Higher power outputs, related to lower ohmic losses, are observed when the membrane is assembled with the lower equivalent weight layer (Aquivion™) at the anode side. This orientation, corresponding to enhanced water transport by back-flow while electroosmosis remains unaffected, results in the higher hydration of the membrane and of the anode active layer during operation. Also, polarization data suggest a different water repartition in the fuel cell along the on-plane direction. Even if the interest in multi-layer PFSA membranes as perspective electrolytes for PEMFCs is not definitively attested, these materials appear to be excellent model systems to establish relationships between the membrane transport properties, the water distribution in the fuel cell and the electrochemical performances. Thanks to the micrometric resolution, in situ Raman microspectroscopy proves to be a unique tool to measure the actual hydration of the membrane at the surface swept by the hydrated feed gases during operation, so that it can be used as a local probe of the water concentration evolution along the gas distribution channels according to changing working conditions.

Interaction between bending and tension forces in bilayer membranes.

PubMed Central

Secomb, T W

1988-01-01

A theoretical analysis is presented of the bending mechanics of a membrane consisting of two tightly-coupled leaflets, each of which shears and bends readily but strongly resists area changes. Structures of this type have been proposed to model biological membranes such as red blood cell membrane. It is shown that when such a membrane is bent, anisotropic components of resultant membrane tension (shear stresses) are induced, even when the tension in each leaflet is isotropic. The induced shear stresses increase as the square of the membrane curvature, and become significant for moderate curvatures (when the radius of curvature is much larger than the distance between the leaflets). This effect has implications for the analysis of shape and deformation of freely suspended and flowing red blood cells. PMID:3224154

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

PubMed

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates. Copyright © 2018 Elsevier B.V. All rights reserved.

Biotinylated lipid bilayer disks as model membranes for biosensor analyses.

PubMed

Lundquist, Anna; Hansen, Søren B; Nordström, Helena; Danielson, U Helena; Edwards, Katarina

2010-10-15

The aim of this study was to investigate the potential of polyethylene glycol (PEG)-stabilized lipid bilayer disks as model membranes for surface plasmon resonance (SPR)-based biosensor analyses. Nanosized bilayer disks that included 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)(2000)] (DSPE-PEG(2000)-biotin) were prepared and structurally characterized by cryo-transmission electron microscopy (cryo-TEM) imaging. The biotinylated disks were immobilized via streptavidin to three different types of sensor chips (CM3, CM4, and CM5) varying in their degree of carboxymethylation and thickness of the dextran matrix. The bilayer disks were found to interact with and bind stably to the streptavidin-coated sensor surfaces. As a first step toward the use of these bilayer disks as model membranes in SPR-based studies of membrane proteins, initial investigations were carried out with cyclooxygenases 1 and 2 (COX 1 and COX 2). Bilayer disks were preincubated with the respective protein and thereafter allowed to interact with the sensor surface. The signal resulting from the interaction was, in both cases, significantly enhanced as compared with the signal obtained when disks alone were injected over the surface. The results of the study suggest that bilayer disks constitute a new and promising type of model membranes for SPR-based biosensor studies. Copyright 2010 Elsevier Inc. All rights reserved.

Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface.

PubMed

Cheng, Sara Y; Chou, George; Buie, Creighton; Vaughn, Mark W; Compton, Campbell; Cheng, Kwan H

2016-03-01

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein-lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions for a dimeric protein on membrane surfaces. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Vesiculation of healthy and defective red blood cells

NASA Astrophysics Data System (ADS)

Li, He; Lykotrafitis, George

2015-07-01

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

Uncovering homo-and hetero-interactions on the cell membrane using single particle tracking approaches

NASA Astrophysics Data System (ADS)

Torreno-Pina, Juan A.; Manzo, Carlo; Garcia-Parajo, Maria F.

2016-03-01

The plasma membrane of eukaryotic cells is responsible for a myriad of functions that regulate cell physiology and plays a crucial role in a multitude of processes that include adhesion, migration, signaling recognition and cell-cell communication. This is accomplished by specific interactions between different membrane components such as lipids and proteins on the lipid bilayer but also through interactions with the underlying cortical actin cytoskeleton on the intracellular side and the glycocalyx matrix in close proximity to the extracellular side. Advanced biophysical techniques, including single particle tracking (SPT) have revealed that the lateral diffusion of molecular components on the plasma membrane represents a landmark manifestation of such interactions. Indeed, by studying changes in the diffusivity of individual membrane molecules, including sub-diffusion, confined diffusion and/or transient arrest of molecules in membrane compartments, it has been possible to gain insight on the nature of molecular interactions and to infer on its functional role for cell response. In this review, we will revise some exciting results where SPT has been crucial to reveal homo- and hetero-interactions on the cell membrane.

Calcium and zinc differentially affect the structure of lipid membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kučerka, Norbert; Dushanov, Ermuhammad; Kholmurodov, Kholmirzo T.

Interactions of calcium (Ca 2+) and zinc (Zn 2+) cations with biomimetic membranes made of dipalmitoylphosphatidylcholine (DPPC) were studied by small angle neutron diffraction (SAND). Experiments show that the structure of these lipid bilayers is differentially affected by the two divalent cations. Initially, both Ca 2+ and Zn 2+ cause DPPC bilayers to thicken, while further increases in Ca 2+ concentration result in the bilayer thinning, eventually reverting to having the same thickness as pure DPPC. The binding of Zn 2+, on the other hand, causes the bilayers to swell to a maximum thickness, and the addition of more Znmore » 2+ does not result in a further thickening of the membrane. Agreement between our results obtained using oriented planar membranes and those from vesicular samples implies that the effect of cations on bilayer thickness is the result of electrostatic interactions, rather than geometrical constraints due to bilayer curvature. This notion is further reinforced by MD simulations. Lastly, the radial distribution functions reveal a strong interaction between Ca 2+ and the phosphate oxygens, while Zn 2+ shows a much weaker binding specificity.« less

Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks

NASA Astrophysics Data System (ADS)

Bayburt, Timothy H.; Sligar, Stephen G.

2002-05-01

The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

Calcium and zinc differentially affect the structure of lipid membranes

DOE PAGES

Kučerka, Norbert; Dushanov, Ermuhammad; Kholmurodov, Kholmirzo T.; ...

2017-03-09

Interactions of calcium (Ca 2+) and zinc (Zn 2+) cations with biomimetic membranes made of dipalmitoylphosphatidylcholine (DPPC) were studied by small angle neutron diffraction (SAND). Experiments show that the structure of these lipid bilayers is differentially affected by the two divalent cations. Initially, both Ca 2+ and Zn 2+ cause DPPC bilayers to thicken, while further increases in Ca 2+ concentration result in the bilayer thinning, eventually reverting to having the same thickness as pure DPPC. The binding of Zn 2+, on the other hand, causes the bilayers to swell to a maximum thickness, and the addition of more Znmore » 2+ does not result in a further thickening of the membrane. Agreement between our results obtained using oriented planar membranes and those from vesicular samples implies that the effect of cations on bilayer thickness is the result of electrostatic interactions, rather than geometrical constraints due to bilayer curvature. This notion is further reinforced by MD simulations. Lastly, the radial distribution functions reveal a strong interaction between Ca 2+ and the phosphate oxygens, while Zn 2+ shows a much weaker binding specificity.« less

Simulations of a Membrane-Anchored Peptide: Structure, Dynamics, and Influence on Bilayer Properties

PubMed Central

Jensen, Morten Ø.; Mouritsen, Ole G.; Peters, Günther H.

2004-01-01

A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged. PMID:15189854

Confocal Raman Microscopy for in Situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles.

PubMed

Kitt, Jay P; Bryce, David A; Minteer, Shelley D; Harris, Joel M

2018-06-05

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this work, we employ in situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayers deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically trapped phospholipid vesicle membranes. Additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.

Role of Transbilayer Distribution of Lipid Molecules on the Structure and Protein-Lipid Interaction of an Amyloidogenic Protein on the Membrane Surface

NASA Astrophysics Data System (ADS)

Cheng, Kwan; Cheng, Sara

We used molecular dynamics simulations to examine the effects of transbilayer distribution of lipid molecules, particularly anionic lipids with negatively charged headgroups, on the structure and binding kinetics of an amyloidogenic protein on the membrane surface and subsequent protein-induced structural disruption of the membrane. Our systems consisted of a model beta-sheet rich dimeric protein absorbed on asymmetric bilayers with neutral and anionic lipids and symmetric bilayers with neutral lipids. We observed larger folding, domain aggregation, and tilt angle of the absorbed protein on the asymmetric bilayer surfaces. We also detected more focused bilayer thinning in the asymmetric bilayer due to weak lipid-protein interactions. Our results support the mechanism that the higher lipid packing in the protein-contacting lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions of an amyloidogenic protein on the membrane surface. We speculate that the observed surface-induced structural and protein-lipid interaction of our model amyloidogenic protein may play a role in the early membrane-associated amyloid cascade pathway that leads to membrane structural damage of neurons in Alzheimer's disease. NSF ACI-1531594.

Interactions of the Auxilin-1 PTEN-like Domain with Model Membranes Result in Nanoclustering of Phosphatidyl Inositol Phosphates

PubMed Central

Kalli, Antreas C.; Morgan, Gareth; Sansom, Mark S.P.

2013-01-01

Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP2) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP2 molecules in the adjacent bilayer leaflet. PMID:23823232

Molecular dynamics exploration of poration and leaking caused by Kalata B1 in HIV-infected cell membrane compared to host and HIV membranes.

PubMed

Nawae, Wanapinun; Hannongbua, Supa; Ruengjitchatchawalya, Marasri

2017-06-15

The membrane disruption activities of kalata B1 (kB1) were investigated using molecular dynamics simulations with membrane models. The models were constructed to mimic the lipid microdomain formation in membranes of HIV particle, HIV-infected cell, and host cell. The differences in the lipid ratios of these membranes caused the formation of liquid ordered (lo) domains of different sizes, which affected the binding and activity of kB1. Stronger kB1 disruptive activity was observed for the membrane with small sized lo domain. Our results show that kB1 causes membrane leaking without bilayer penetration. The membrane poration mechanism involved in the disorganization of the lo domain and in cholesterol inter-leaflet translocation is described. This study enhances our understanding of the membrane activity of kB1, which may be useful for designing novel and potentially therapeutic peptides based on the kB1 framework.

Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes.

PubMed

Becucci, Lucia; Papini, Martina; Mullen, Daniel; Scaloni, Andrea; Veglia, Gianluigi; Guidelli, Rolando

2011-11-01

The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side. Copyright © 2011 Elsevier B.V. All rights reserved.

Smart polymer brush nanostructures guide the self-assembly of pore-spanning lipid bilayers with integrated membrane proteins

NASA Astrophysics Data System (ADS)

Wilhelmina de Groot, G.; Demarche, Sophie; Santonicola, M. Gabriella; Tiefenauer, Louis; Vancso, G. Julius

2014-01-01

Nanopores in arrays on silicon chips are functionalized with pH-responsive poly(methacrylic acid) (PMAA) brushes and used as supports for pore-spanning lipid bilayers with integrated membrane proteins. Robust platforms are created by the covalent grafting of polymer brushes using surface-initiated atom transfer radical polymerization (ATRP), resulting in sensor chips that can be successfully reused over several assays. His-tagged proteins are selectively and reversibly bound to the nitrilotriacetic acid (NTA) functionalization of the PMAA brush, and consequently lipid bilayer membranes are formed. The enhanced membrane resistance as determined by electrochemical impedance spectroscopy and free diffusion of dyed lipids observed as fluorescence recovery after photobleaching confirmed the presence of lipid bilayers. Immobilization of the His-tagged membrane proteins on the NTA-modified PMAA brush near the pore edges is characterized by fluorescence microscopy. This system allows us to adjust the protein density in free-standing bilayers, which are stabilized by the polymer brush underneath. The potential application of the integrated platform for ion channel protein assays is demonstrated.

Thermal and active fluctuations of a compressible bilayer vesicle

NASA Astrophysics Data System (ADS)

Sachin Krishnan, T. V.; Yasuda, Kento; Okamoto, Ryuichi; Komura, Shigeyuki

2018-05-01

We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.

Molecular Dynamics Simulation and Analysis of the Antimicrobial Peptide-Lipid Bilayer Interactions.

PubMed

Arasteh, Shima; Bagheri, Mojtaba

2017-01-01

A great deal of research has been undertaken in order to discover antimicrobial peptides (AMPs) with unexploited mechanisms of action to counteract the health-threatening issues associated with bacterial resistance. The intrinsic effectiveness of AMPs is strongly influenced by their initial interactions with the bacterial cell membrane. Understanding these interactions in the atomistic details is important for the design of the less prone bacteria-resistant peptides. However, these studies always require labor-intensive and difficult steps. With this regard, modeling studies of the AMPs binding to simple lipid membrane systems, e.g., lipid bilayers, is of great advantage. In this chapter, we present an applicable step-by-step protocol to run the molecular dynamics (MD) simulation of the interaction between cyclo-RRWFWR (c-WFW) (a small cyclic AMP) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer using the Groningen machine for chemical simulations (GROMACS) package. The protocol as described here may simply be optimized for other peptide-lipid systems of interest.

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.

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

PubMed

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

2016-08-26

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.

Modeling of interactions between nanoparticles and cell membranes

NASA Astrophysics Data System (ADS)

Ban, Young-Min

Rapid development of nanotechnology and ability to manufacture materials and devices with nanometer feature size leads to exciting innovations in many areas including the medical and electronic fields. However, the possible health and environmental impacts of manufactured nanomaterials are not fully known. Recent experimental reports suggest that some of the manufactured nanomaterials, such as fullerenes and carbon nanotubes, are highly toxic even in small concentrations. The goal of the current work is to understand the mechanisms responsible for the toxicity of nanomaterials. In the current study coarse-grained molecular dynamics simulations are employed to investigate the interactions between NPs and cellular membranes at a molecular level. One of the possible toxicity mechanisms of the nanomaterials is membrane disruption. Possibility of membrane disruption exposed to the manufactured nanomaterials are examined by considering chemical reactions and non-reactive physical interactions as chemical as well as physical mechanisms. Mechanisms of transport of carbon-based nanoparticles (fullerene and its derivative) across a phospholipid bilayer are investigated. The free energy profile is obtained using constrained simulations. It is shown that the considered nanoparticles are hydrophobic and therefore they tend to reside in the interior of the lipid bilayer. In addition, the dynamics of the membrane fluctuations is significantly affected by the nanoparticles at the bilayer-water interface. The hydrophobic interaction between the particles and membrane core induces the strong coupling between the nanoparticle motion and membrane deformation. It is observed that the considered nanoparticles affect several physical properties of the membrane. The nanoparticles embedded into the membrane interior lead to the membrane softening, which becomes more significant with increase in CNT length and concentration. The lateral pressure profile and membrane energy in the membrane containing the nanoparticles exhibit localized perturbation around the nanoparticle. The nanoparticles are not likely to affect membrane protein function by the weak perturbation of the internal stress in the membrane. Due to the short-ranged interactions between the nanoparticles, the nanoparticles would not form aggregates inside membranes. The effect of lipid peroxidation on cell membrane deformation is assessed. The peroxidized lipids introduce a perturbation to the internal structure of the membrane leading to higher amplitude of the membrane fluctuations. Higher concentration of the peroxidized lipids induces more significant perturbation. Cumulative effects of lipid peroxidation caused by nanoparticles are examined for the first time. The considered amphiphilic particle appears to reduce the perturbation of the membrane structure at its equilibrium position inside the peroxidized membrane. This suggests a possibility of antioxidant effect of the nanoparticle.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes.

PubMed

Tiriveedhi, Venkataswarup; Kitchens, Kelly M; Nevels, Kerrick J; Ghandehari, Hamidreza; Butko, Peter

2011-01-01

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern-Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells. Copyright © 2010 Elsevier B.V. All rights reserved.

Monovalent Ions and Water Dipoles in Contact with Dipolar Zwitterionic Lipid Headgroups-Theory and MD Simulations

PubMed Central

Velikonja, Aljaž; Perutkova, Šarka; Gongadze, Ekaterina; Kramar, Peter; Polak, Andraž; Maček-Lebar, Alenka; Iglič, Aleš

2013-01-01

The lipid bilayer is a basic building block of biological membranes and can be pictured as a barrier separating two compartments filled with electrolyte solution. Artificial planar lipid bilayers are therefore commonly used as model systems to study the physical and electrical properties of the cell membranes in contact with electrolyte solution. Among them the glycerol-based polar phospholipids which have dipolar, but electrically neutral head groups, are most frequently used in formation of artificial lipid bilayers. In this work the electrical properties of the lipid layer composed of zwitterionic lipids with non-zero dipole moments are studied theoretically. In the model, the zwitterionic lipid bilayer is assumed to be in contact with aqueous solution of monovalent salt ions. The orientational ordering of water, resulting in spatial variation of permittivity, is explicitly taken into account. It is shown that due to saturation effect in orientational ordering of water dipoles the relative permittivity in the zwitterionic headgroup region is decreased, while the corresponding electric potential becomes strongly negative. Some of the predictions of the presented mean-field theoretical consideration are critically evaluated using the results of molecular dynamics (MD) simulation. PMID:23434651

A Neutron View of Proteins in Lipid Bilayers

NASA Astrophysics Data System (ADS)

White, Stephen

2012-02-01

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly-charged S1-S4 voltage- sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated potassium channels. We have used neutron diffraction, solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field.

Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic

PubMed Central

Clifton, Luke A.; Skoda, Maximilian W. A.; Daulton, Emma L.; Hughes, Arwel V.; Le Brun, Anton P.; Lakey, Jeremy H.; Holt, Stephen A.

2013-01-01

The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions. PMID:24132206

Selective effect of cell membrane on synaptic neurotransmission

NASA Astrophysics Data System (ADS)

Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

2016-01-01

Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

Primate cathelicidin orthologues display different structures and membrane interactions.

PubMed

Morgera, Francesca; Vaccari, Lisa; Antcheva, Nikolinka; Scaini, Denis; Pacor, Sabrina; Tossi, Alessandro

2009-02-01

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.

Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

PubMed

Kreidler, Anna-Maria; Benz, Roland; Barth, Holger

2017-03-01

The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B 7 ) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B 7 components of C2 toxin and LT, C2IIa and PA 63 , respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA 63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA 63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA 63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA 63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

Membrane-Surface Anchoring of Charged Diacylglycerol-Lactones Correlates with Biological Activities | Center for Cancer Research

Cancer.gov

The inside cover picture shows the molecular structure of a DAG lactone derivative on top of the inner leaflet of a DMPC bilayer. The confocal microscopy image illustrates DAG-lactone-stimulated membrane localization of PKCδ-ECFP in living cells, while the space-filling model shows the surface of the C1B domain of PKCδ, the target of the lactone.

Effect of plasma membrane fluidity on serotonin transport by endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Block, E.R.; Edwards, D.

1987-11-01

To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotinin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of ({sup 14}C)-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluiditymore » in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect of fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells.« less

The nanoscale organization of the B lymphocyte membrane☆

PubMed Central

Maity, Palash Chandra; Yang, Jianying; Klaesener, Kathrin; Reth, Michael

2015-01-01

The fluid mosaic model of Singer and Nicolson correctly predicted that the plasma membrane (PM) forms a lipid bi-layer containing many integral trans-membrane proteins. This model also suggested that most of these proteins were randomly dispersed and freely diffusing moieties. Initially, this view of a dynamic and rather unorganized membrane was supported by early observations of the cell surfaces using the light microscope. However, recent studies on the PM below the diffraction limit of visible light (~ 250 nm) revealed that, at nanoscale dimensions, membranes are highly organized and compartmentalized structures. Lymphocytes are particularly useful to study this nanoscale membrane organization because they grow as single cells and are not permanently engaged in cell:cell contacts within a tissue that can influence membrane organization. In this review, we describe the methods that can be used to better study the protein:protein interaction and nanoscale organization of lymphocyte membrane proteins, with a focus on the B cell antigen receptor (BCR). Furthermore, we discuss the factors that may generate and maintain these membrane structures. PMID:25450974

Interaction of saponin 1688 with phase separated lipid bilayers.

PubMed

Chen, Maohui; Balhara, Vinod; Jaimes Castillo, Ana Maria; Balsevich, John; Johnston, Linda J

2017-07-01

Saponins are a diverse family of naturally occurring plant triterpene or steroid glycosides that have a wide range of biological activities. They have been shown to permeabilize membranes and in some cases membrane disruption has been hypothesized to involve saponin/cholesterol complexes. We have examined the interaction of steroidal saponin 1688-1 with lipid membranes that contain cholesterol and have a mixture of liquid-ordered (L o ) and liquid-disordered (L d ) phases as a model for lipid rafts in cellular membranes. A combination of atomic force microscopy (AFM) and fluorescence was used to probe the effect of saponin on the bilayer. The results demonstrate that saponin forms defects in the membrane and also leads to formation of small aggregates on the membrane surface. Although most of the membrane damage occurs in the liquid-disordered phase, fluorescence results demonstrate that saponin localizes in both ordered and disordered membrane phases, with a modest preference for the disordered regions. Similar effects are observed for both direct incorporation of saponin in the lipid mixture used to make vesicles/bilayers and for incubation of saponin with preformed bilayers. The results suggest that the initial sites of interaction are at the interface between the domains and surrounding disordered phase. The preference for saponin localization in the disordered phase may reflect the ease of penetration of saponin into a less ordered membrane, rather than the actual cholesterol concentration in the membrane. Dye leakage assays indicate that a high concentration of saponin is required for membrane permeabilization consistent with the supported lipid bilayer experiments. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

A two-helix motif positions the active site of lysophosphatidic acid acyltransferase for catalysis within the membrane bilayer

PubMed Central

Robertson, Rosanna M.; Yao, Jiangwei; Gajewski, Stefan; Kumar, Gyanendra; Martin, Erik W.; Rock, Charles O.; White, Stephen W.

2017-01-01

Phosphatidic acid is the central intermediate in membrane phospholipid synthesis and is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycero-3-phosphate acyltransferase, called PlsC in bacteria. The crystal structure of PlsC from Thermotoga maritima reveals an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity ‘ruler’ is an appropriately placed and closed hydrophobic tunnel. This was confirmed by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells expressing the mutated proteins. MD simulations reveal that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer using soluble acyl donors. PMID:28714993

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

PubMed Central

Lehto, M T; Sharom, F J

1998-01-01

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane. PMID:9576857

Cardiolipin effects on membrane structure and dynamics.

PubMed

Unsay, Joseph D; Cosentino, Katia; Subburaj, Yamunadevi; García-Sáez, Ana J

2013-12-23

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.

Differential affinities of MinD and MinE to anionic phospholipid influence Min Patterning dynamics in vitro

PubMed Central

Vecchiarelli, Anthony G.; Li, Min; Mizuuchi, Michiyo; Mizuuchi, Kiyoshi

2014-01-01

The E. coli Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid-cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no-flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD-independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning. PMID:24930948

Diffusion in phospholipid bilayer membranes: dual-leaflet dynamics and the roles of tracer–leaflet and inter-leaflet coupling

PubMed Central

Hill, Reghan J.; Wang, Chih-Ying

2014-01-01

A variety of observations—sometimes controversial—have been made in recent decades when attempting to elucidate the roles of interfacial slip on tracer diffusion in phospholipid membranes. Evans–Sackmann theory (1988) has furnished membrane viscosities and lubrication-film thicknesses for supported membranes from experimentally measured lateral diffusion coefficients. Similar to the Saffman and Delbrück model, which is the well-known counterpart for freely supported membranes, the bilayer is modelled as a single two-dimensional fluid. However, the Evans–Sackman model cannot interpret the mobilities of monotopic tracers, such as individual lipids or rigidly bound lipid assemblies; neither does it account for tracer–leaflet and inter-leaflet slip. To address these limitations, we solve the model of Wang and Hill, in which two leaflets of a bilayer membrane, a circular tracer and supports are coupled by interfacial friction, using phenomenological friction/slip coefficients. This furnishes an exact solution that can be readily adopted to interpret the mobilities of a variety of mosaic elements—including lipids, integral monotopic and polytopic proteins, and lipid rafts—in supported bilayer membranes. PMID:25002822

Controlling the shape of membrane protein polyhedra

NASA Astrophysics Data System (ADS)

Li, Di; Kahraman, Osman; Haselwandter, Christoph A.

2017-03-01

Membrane proteins and lipids can self-assemble into membrane protein polyhedral nanoparticles (MPPNs). MPPNs have a closed spherical surface and a polyhedral protein arrangement, and may offer a new route for structure determination of membrane proteins and targeted drug delivery. We develop here a general analytic model of how MPPN self-assembly depends on bilayer-protein interactions and lipid bilayer mechanical properties. We find that the bilayer-protein hydrophobic thickness mismatch is a key molecular control parameter for MPPN shape that can be used to bias MPPN self-assembly towards highly symmetric and uniform MPPN shapes. Our results suggest strategies for optimizing MPPN shape for structural studies of membrane proteins and targeted drug delivery.

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

PubMed

Saheki, Yasunori; De Camilli, Pietro

2017-06-20

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

Self-Assembly of Phosphate Amphiphiles in Mixtures of Prebiotically Plausible Surfactants

PubMed Central

Albertsen, A.N.; Duffy, C.D.; Sutherland, J.D.

2014-01-01

Abstract The spontaneous formation of closed bilayer structures from prebiotically plausible amphiphiles is an essential requirement for the emergence of early cells on prebiotic Earth. The sources of amphiphiles could have been both endo- and exogenous (accretion of meteorite carbonaceous material or interstellar dust particles). Among all prebiotic possible amphiphile candidates, those containing phosphate are the least investigated species because their self-assembly occurs in a seemingly too narrow range of conditions. The self-assembly of simple phosphate amphiphiles should, however, be of great interest, as contemporary membranes predominantly contain phospholipids. In contrast to common expectations, we show that these amphiphiles can be easily synthesized under prebiotically plausible environmental conditions and can efficiently form bilayer structures in the presence of various co-surfactants across a large range of pH values. Vesiculation was even observed in crude reaction mixtures that contained 1-decanol as the amphiphile precursor. The two best co-surfactants promoted vesicle formation over the entire pH range in aqueous solutions. Expanding the pH range where bilayer membranes self-assemble and remain intact is a prerequisite for the emergence of early cell-like compartments and their preservation under fluctuating environmental conditions. These mixed bilayers also retained small charged solutes, such as dyes. These results demonstrate that alkyl phosphate amphiphiles might have played a significant role as early compartment building blocks. Key Words: Vesicles—Alkyl phosphate—Prebiotic synthesis—Amphiphile mixtures. Astrobiology 14, 462–472. PMID:24885934

Mammalian phospholipid homeostasis: evidence that membrane curvature elastic stress drives homeoviscous adaptation in vivo

PubMed Central

2016-01-01

Several theories of phospholipid homeostasis have postulated that cells regulate the molecular composition of their bilayer membranes, such that a common biophysical membrane parameter is under homeostatic control. Two commonly cited theories are the intrinsic curvature hypothesis, which states that cells control membrane curvature elastic stress, and the theory of homeoviscous adaptation, which postulates cells control acyl chain packing order (membrane order). In this paper, we present evidence from data-driven modelling studies that these two theories correlate in vivo. We estimate the curvature elastic stress of mammalian cells to be 4–7 × 10−12 N, a value high enough to suggest that in mammalian cells the preservation of membrane order arises through a mechanism where membrane curvature elastic stress is controlled. These results emerge from analysing the molecular contribution of individual phospholipids to both membrane order and curvature elastic stress in nearly 500 cellular compositionally diverse lipidomes. Our model suggests that the de novo synthesis of lipids is the dominant mechanism by which cells control curvature elastic stress and hence membrane order in vivo. These results also suggest that cells can increase membrane curvature elastic stress disproportionately to membrane order by incorporating polyunsaturated fatty acids into lipids. PMID:27534697

Mammalian phospholipid homeostasis: evidence that membrane curvature elastic stress drives homeoviscous adaptation in vivo.

PubMed

Dymond, Marcus K

2016-08-01

Several theories of phospholipid homeostasis have postulated that cells regulate the molecular composition of their bilayer membranes, such that a common biophysical membrane parameter is under homeostatic control. Two commonly cited theories are the intrinsic curvature hypothesis, which states that cells control membrane curvature elastic stress, and the theory of homeoviscous adaptation, which postulates cells control acyl chain packing order (membrane order). In this paper, we present evidence from data-driven modelling studies that these two theories correlate in vivo. We estimate the curvature elastic stress of mammalian cells to be 4-7 × 10(-12) N, a value high enough to suggest that in mammalian cells the preservation of membrane order arises through a mechanism where membrane curvature elastic stress is controlled. These results emerge from analysing the molecular contribution of individual phospholipids to both membrane order and curvature elastic stress in nearly 500 cellular compositionally diverse lipidomes. Our model suggests that the de novo synthesis of lipids is the dominant mechanism by which cells control curvature elastic stress and hence membrane order in vivo These results also suggest that cells can increase membrane curvature elastic stress disproportionately to membrane order by incorporating polyunsaturated fatty acids into lipids. © 2016 The Author(s).

The influence of a non-occlusive bi-layer composite membrane on skin barrier properties. A non-invasive evaluation with a right-left intra-individual pre/post comparison study.

PubMed

Brazzelli, V; Berardesca, E; Rona, C; Borroni, G

2008-01-01

The purpose of this placebo-controlled right-left intra-individual pre/post comparison study was to evaluate the efficacy of a new bi-layer composite membrane, composed of a layer of knitted cotton and a layer of semi-permeable polyurethane, developed in order to improve skin hydration. Eighteen healthy subjects entered the study. A T-shirt, dedicated to this study, was prepared and it was worn for 8 h, mimicking overnight wearing. Before and at the removal of the T-shirt an objective quantification of skin parameters was performed by measuring hydration, transepidermal water loss (TEWL) and skin surface pH, bilaterally, on the inner side of the forearm. Measurements were performed both at the interface between the skin and the bi-layer composite membrane or cotton and on the outer side of the membrane (to assess permeation of water and occlusive properties of the product) with and without a single application of a moisturizer. A statistically significant improvement of skin hydration, recorded on the stratum corneum underneath the bi-layer membrane versus cotton alone, was measured both with (p < 0.0001) and without application of the moisturizer (p < 0.002). TEWL was shown to decrease significantly on the side of the bi-layer membrane, if compared with cotton (p < 0.008), after application of the moisturizer. TEWL through the membrane showed no significant differences as compared to placebo, confirming the permeability of the fabric. Our data suggest that this bi-layer composite membrane can promote the hydration process of the stratum corneum, increasing the hydrating properties of the moisturizer agent. (c) 2007 S. Karger AG, Basel

Breathable NIPAAm Network with Controllable Hydration Supports Model Lipid Membrane

NASA Astrophysics Data System (ADS)

Jablin, Michael; Smith, Hillary; Zhernenkov, Mikhail; Vidyasagar, Ajay; Toomey, Ryan; Saiz, Jessica; Toperverg, Boris; Watkins, Erik; Kuhl, Tonya; Hurd, Alan; Majewski, Jaroslaw

2009-03-01

The interaction of a model lipid bilayer composed of DPPC with a surface-tethered poly(N-isopropylacrylamide) (NIPAAm) was explored with neutron reflectometry (NR). The Langmuir-Blodgett / Langmuir-Schaeffer method was used to deposit a lipid bilayer onto the polymer. NR measurements were used to probe the in- and out-of-plane structure of the system as a function of temperature. NR with fluorescence microscopy show that the polymer supports a lipid bilayer, and hydration of the support can be controlled. At low temp. the membrane develops out-of-plane undulations visible in off-specular scattering. Analysis of the off-specular reveals in-plane correlation of the bilayer fluctuations. The separation of the lipid bilayer from the solid support of a substrate constitutes a significant step towards a more realistic model of biological membranes.

Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rai, Durgesh K.; Qian, Shuo

Aurein 1.2 is a potent antimicrobial peptide secreted by frog Litoria aurea. As a short membrane-active peptide with only 13 amino acids in sequence, it has been found to be residing on the surface of lipid bilayer and permeabilizing bacterial membranes at high concentration. However, the detail at the molecular level is largely unknown. Here in this study, we investigated the action of Aurein 1.2 in charged lipid bilayers composed of DMPC/DMPG. Oriented Circular Dichroism results showed that the peptide was on the surface of lipid bilayer regardless of the charged lipid ratio. Only at a very high peptide-to-lipid ratiomore » (~1/10), the peptide became perpendicular to the bilayer, however no pore was detected by neutron in-plane scattering. To further understand how it interacted with charged lipid bilayers, we employed Small Angle Neutron Scattering to probe lipid distribution across bilayer leaflets in lipid vesicles. The results showed that Aurein 1.2 interacted strongly with negatively charged DMPG, causing strong asymmetry in lipid bilayer. At high concentration, while the vesicles were intact, we found additional structure feature on the bilayer. Finally, our study provides a glimpse into how Aurein 1.2 disturbs anionic lipid-containing membranes without pore formation.« less

Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer

DOE PAGES

Rai, Durgesh K.; Qian, Shuo

2017-06-16

Aurein 1.2 is a potent antimicrobial peptide secreted by frog Litoria aurea. As a short membrane-active peptide with only 13 amino acids in sequence, it has been found to be residing on the surface of lipid bilayer and permeabilizing bacterial membranes at high concentration. However, the detail at the molecular level is largely unknown. Here in this study, we investigated the action of Aurein 1.2 in charged lipid bilayers composed of DMPC/DMPG. Oriented Circular Dichroism results showed that the peptide was on the surface of lipid bilayer regardless of the charged lipid ratio. Only at a very high peptide-to-lipid ratiomore » (~1/10), the peptide became perpendicular to the bilayer, however no pore was detected by neutron in-plane scattering. To further understand how it interacted with charged lipid bilayers, we employed Small Angle Neutron Scattering to probe lipid distribution across bilayer leaflets in lipid vesicles. The results showed that Aurein 1.2 interacted strongly with negatively charged DMPG, causing strong asymmetry in lipid bilayer. At high concentration, while the vesicles were intact, we found additional structure feature on the bilayer. Finally, our study provides a glimpse into how Aurein 1.2 disturbs anionic lipid-containing membranes without pore formation.« less

Tip-enhanced Raman spectroscopy of lipid bilayers in water with an alumina- and silver-coated tungsten tip.

PubMed

Nakata, Atsushi; Nomoto, Tomonori; Toyota, Taro; Fujinami, Masanori

2013-01-01

Tip-enhanced Raman spectroscopy (TERS) of supported phospholipid bilayers in an aqueous environment is discussed in this paper. Two bilayer membranes were examined: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We fabricated alumina- and silver-coated tungsten tips that are very robust in water. There was a large time-dependence in the TERS spectra for the DOPC bilayers, whereas no such time-dependence was observed in the DPPC bilayer spectra under the probe tip. The spectral changes of DOPC bilayers are discussed in terms of the fluidity of the liquid crystalline phase. Time-resolved TERS thus has the potential to characterize inhomogeneity and diffusion in fluidic phospholipid bilayer membranes.

Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack.

PubMed

van der Goot, F G; Harder, T

2001-04-01

While the existence of cholesterol/sphingolipid (raft) membrane domains in the plasma membrane is now supported by strong experimental evidence, the structure of these domains, their size, their dynamics, and their molecular composition remain to be understood. Raft domains are thought to represent a specific physical state of lipid bilayers, the liquid-ordered phase. Recent observations suggest that in the mammalian plasma membrane small raft domains in ordered lipid phases are in a dynamic equilibrium with a less ordered membrane environment. Rafts may be enlarged and/or stabilized by protein-mediated cross-linking of raft-associated components. These changes of plasma membrane structure are perceived by the cells as signals, most likely an important element of immunoreceptor signalling. Pathogens abuse raft domains on the host cell plasma membrane as concentration devices, as signalling platforms and/or entry sites into the cell. Elucidation of these interactions requires a detailed understanding raft structure and dynamics. Copyright 2001 Academic Press.

Fusion of raft-like lipid bilayers operated by a membranotropic domain of the HSV-type I glycoprotein gH occurs through a cholesterol-dependent mechanism.

PubMed

Vitiello, Giuseppe; Falanga, Annarita; Petruk, Ariel Alcides; Merlino, Antonello; Fragneto, Giovanna; Paduano, Luigi; Galdiero, Stefania; D'Errico, Gerardino

2015-04-21

A wealth of evidence indicates that lipid rafts are involved in the fusion of the viral lipid envelope with the target cell membrane. However, the interplay between these sterol- and sphingolipid-enriched ordered domains and viral fusion glycoproteins has not yet been clarified. In this work we investigate the molecular mechanism by which a membranotropic fragment of the glycoprotein gH of the Herpes Simplex Virus (HSV) type I (gH625) drives fusion of lipid bilayers formed by palmitoyl oleoyl phosphatidylcholine (POPC)-sphingomyelin (SM)-cholesterol (CHOL) (1 : 1 : 1 wt/wt/wt), focusing on the role played by each component. The comparative analysis of the liposome fusion assays, Dynamic Light Scattering (DLS), spectrofluorimetry, Neutron Reflectivity (NR) and Electron Spin Resonance (ESR) experiments, and Molecular Dynamics (MD) simulations shows that CHOL is fundamental for liposome fusion to occur. In detail, CHOL stabilizes the gH625-bilayer association by specific interactions with the peptide Trp residue. The interaction with gH625 causes an increased order of the lipid acyl chains, whose local rotational motion is significantly hampered. SM plays only a minor role in the process, favoring the propagation of lipid perturbation to the bilayer inner core. The stiffening of the peptide-interacting bilayer leaflet results in an asymmetric perturbation of the membrane, which is locally destabilized thus favoring fusion events. Our results show that viral fusion glycoproteins are optimally suited to exert a high fusogenic activity on lipid rafts and support the relevance of cholesterol as a key player of membrane-related processes.

Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

PubMed Central

Fuks, B; Homblé, F

1994-01-01

Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes. PMID:8061192

Reversible Lifting of Surface Supported Lipid Bilayers with a Membrane-Spanning Nonionic Triblock Copolymer

DOE PAGES

Hayden, Steven C.; Junghans, Ann; Majewski, Jaroslaw; ...

2017-02-22

Neutron reflectometry was used to monitor structural variations in surface supported DMPC bilayers induced by the addition of Triton X-100, a surfactant commonly used to aid solubilization of membrane proteins, and the co-addition of a membrane spanning non-ionic amphiphilic triblock copolymer, (PEO 117-PPO 47-PE O117, Pluronic F98). Surfactant addition causes slight compression of the bilayer thickness and the creation of a distinct EO layer that increases the hydrophilic layer proximal to the supporting substrate (i.e., a water and EO gap between the lipid bilayer and quartz) to 6.8 ± 0.4 Å. Addition of the triblock copolymer into the DMPC: Tritonmore » X-100 bilayer increases the complexity (broadens) the lipid phase transition, further compresses the bilayer, and continues to expand the proximal hydrophilic layer thickness. The observed structural changes are temperature dependent with transmembrane polymer insertion achieved at 37 °C leading to a compressed membrane thickness of 39.2 ± 0.2 Å and proximal gap of 45.2 ± 0.2 Å. Temperature driven exclusion of the polymer at 15 °C causes partitioning of the polymer into the proximal space generating a large hydrogel cushion 162 ± 16 Å thick. An intermediate gap width (10 – 27 Å) is achieved at room temperature (22 – 25 °C). The temperature-driven changes in the proximal hydrophilic gap dimensions are shown to be reversible but thermal history causes variation in magnitude. Temperature-driven changes in polymer association with a supported lipid bilayer offer a facile means to reversibly control both the membrane characteristics as well as the separation between membrane and solid substrate.« less

Kinetic Defects Induced by Melittin in Model Lipid Membranes: A Solution Atomic Force Microscopy Study.

PubMed

Pan, Jianjun; Khadka, Nawal K

2016-05-26

Quantitative characterization of membrane defects (pores) is important for elucidating the molecular basis of many membrane-active peptides. We study kinetic defects induced by melittin in vesicular and planar lipid bilayers. Fluorescence spectroscopy measurements indicate that melittin induces time-dependent calcein leakage. Solution atomic force microscopy (AFM) is used to visualize melittin-induced membrane defects. After initial equilibration, the most probable defect radius is ∼3.8 nm in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) bilayers. Unexpectedly, defects become larger with longer incubation, accompanied by substantial shape transformation. The initial defect radius is ∼4.7 nm in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. Addition of 30 mol % cholesterol to DOPC bilayers suppresses defect kinetics, although the inhibitory impact is negated by longer incubation. Overall, the kinetic rate of defect development follows DLPC > DOPC > DOPC/cholesterol. Kinetic defects are also observed when anionic lipids are present. Based on the observation that defects can occupy as large as 40% of the bilayer surface, we propose a kinetic defect growth model. We also study the effect of melittin on the phase behavior of DOPC/egg-sphingomyelin/cholesterol bilayers. We find that melittin initially suppresses or eliminates liquid-ordered (Lo) domains; Lo domains gradually emerge and become the dominant species with longer incubation; and defects in phase-coexisting bilayers have a most probable radius of ∼5 nm and are exclusively localized in the liquid-disordered (Ld) phase. Our experimental data highlight that melittin-induced membrane defects are not static; conversely, spontaneous defect growth is intrinsically associated with membrane permeabilization exerted by melittin.

Reversible Lifting of Surface Supported Lipid Bilayers with a Membrane-Spanning Nonionic Triblock Copolymer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayden, Steven C.; Junghans, Ann; Majewski, Jaroslaw

Neutron reflectometry was used to monitor structural variations in surface supported DMPC bilayers induced by the addition of Triton X-100, a surfactant commonly used to aid solubilization of membrane proteins, and the co-addition of a membrane spanning non-ionic amphiphilic triblock copolymer, (PEO 117-PPO 47-PE O117, Pluronic F98). Surfactant addition causes slight compression of the bilayer thickness and the creation of a distinct EO layer that increases the hydrophilic layer proximal to the supporting substrate (i.e., a water and EO gap between the lipid bilayer and quartz) to 6.8 ± 0.4 Å. Addition of the triblock copolymer into the DMPC: Tritonmore » X-100 bilayer increases the complexity (broadens) the lipid phase transition, further compresses the bilayer, and continues to expand the proximal hydrophilic layer thickness. The observed structural changes are temperature dependent with transmembrane polymer insertion achieved at 37 °C leading to a compressed membrane thickness of 39.2 ± 0.2 Å and proximal gap of 45.2 ± 0.2 Å. Temperature driven exclusion of the polymer at 15 °C causes partitioning of the polymer into the proximal space generating a large hydrogel cushion 162 ± 16 Å thick. An intermediate gap width (10 – 27 Å) is achieved at room temperature (22 – 25 °C). The temperature-driven changes in the proximal hydrophilic gap dimensions are shown to be reversible but thermal history causes variation in magnitude. Temperature-driven changes in polymer association with a supported lipid bilayer offer a facile means to reversibly control both the membrane characteristics as well as the separation between membrane and solid substrate.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Jianjun; Cheng, Xiaolin; Heberle, Frederick A

Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol s molecular interactions with ether lipids as determined using a combination of small-angle neutron and Xray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data.more » This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup s phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid-mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules.« less

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

PubMed Central

Chester, D W; Herbette, L G; Mason, R P; Joslyn, A F; Triggle, D J; Koppel, D E

1987-01-01

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:2447967

Confocal Raman Microscopy for In-situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kitt, Jay P.; Bryce, David A.; Minteer, Shelley D.

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this paper, we employ in-situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayersmore » deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically-trapped phospholipid vesicle membranes. Finally and additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.« less

Confocal Raman Microscopy for In-situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles

DOE PAGES

Kitt, Jay P.; Bryce, David A.; Minteer, Shelley D.; ...

2018-05-14

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this paper, we employ in-situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayersmore » deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically-trapped phospholipid vesicle membranes. Finally and additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.« less

A Monomeric Membrane Peptide that Lives in Three Worlds: In Solution, Attached to, and Inserted across Lipid Bilayers

PubMed Central

Reshetnyak, Yana K.; Segala, Michael; Andreev, Oleg A.; Engelman, Donald M.

2007-01-01

The membrane peptide pH (low) insertion peptide (pHLIP) lives in three worlds, being soluble in aqueous solution at pH 7.4, binding to the surface of lipid bilayers, and inserting as a transbilayer helix at low pH. With low pH driving the process, pHLIP can translocate cargo molecules attached to its C-terminus via a disulfide and release them in the cytoplasm of a cell. Here we examine a key aspect of the mechanism, showing that pHLIP is monomeric in each of its three major states: soluble in water near neutral pH (state I), bound to the surface of a membrane near neutral pH (state II), and inserted across the membrane as an α-helix at low pH (state III). The peptide does not induce fusion or membrane leakage. The unique properties of pHLIP made it attractive for the biophysical investigation of membrane protein folding in vitro and for the development of a novel class of delivery peptides for the transport of therapeutic and diagnostic agents to acidic tissue sites associated with various pathological processes in vivo. PMID:17557792

Influence of electric field on the amyloid-β(29-42) peptides embedded in a membrane bilayer

NASA Astrophysics Data System (ADS)

Lu, Yan; Shi, Xiao-Feng; Salsbury, Freddie R.; Derreumaux, Philippe

2018-01-01

Alzheimer's disease is linked to various types of aggregates of amyloid-β (Aβ) peptide and their interactions with protein receptors and neuronal cell membranes. Little is known on the impact of the electric field on membrane-embedded Aβ. Here we use atomistic molecular dynamics simulations to study the effects of a constant electric field on the conformations of Aβ29-42 dimer inside a membrane, where the electric field has a strength of 20 mV/nm which exists across the membrane of a human neuron. Starting from α-helix peptides, the transmembrane electric field (TMEF) accelerates the conversion from the Gly-out substate to the Gly-side and Gly-in substates. Starting from β-sheet peptides, TMEF induces changes of the kink and tilt angles at Gly33 and Gly37. Overall, in the simulations totaling 10 μs, TMEF establishes new ground states for the dimer, similar to induced-fit in ligand binding. Our findings indicate that TMEF can stabilize rare conformations of amyloid peptides, and this could influence the cleavage of the amyloid precursor protein and the formation of β-sheet oligomers in membrane bilayers.

Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L.).

PubMed

Brogden, Graham; Propsting, Marcus; Adamek, Mikolaj; Naim, Hassan Y; Steinhagen, Dieter

2014-03-01

Cell membranes act as an interface between the interior of the cell and the exterior environment and facilitate a range of essential functions including cell signalling, cell structure, nutrient uptake and protection. It is composed of a lipid bilayer with integrated proteins, and the inner leaflet of the lipid bilayer comprises of liquid ordered (Lo) and liquid disordered (Ld) domains. Lo microdomains, also named as lipid rafts are enriched in cholesterol, sphingomyelin and certain types of proteins, which facilitate cell signalling and nutrient uptake. Lipid rafts have been extensively researched in mammals and the presence of functional lipid rafts was recently demonstrated in goldfish, but there is currently very little knowledge about their composition and function in fish. Therefore a protocol was established for the analysis of lipid rafts and membranous lipids in common carp (Cyprinus carpio L.) tissues. Twelve lipids were identified and analysed in the Ld domain of the membrane with the most predominant lipids found in all tissues being; triglycerides, cholesterol, phosphoethanolamine and phosphatidylcholine. Four lipids were identified in lipid rafts in all tissues analysed, triglycerides (33-62%) always found in the highest concentration followed by cholesterol (24-32%), phosphatidylcholine and sphingomyelin. Isolation of lipid rafts was confirmed by identifying the presence of the lipid raft associated protein flotillin, present at higher concentrations in the detergent resistant fraction. The data provided here build a lipid library of important carp tissues as a baseline for further studies into virus entry, protein trafficking or environmental stress analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

Particle-based simulations of bilayer membranes: self-assembly, structural analysis, and shock-wave damage

NASA Astrophysics Data System (ADS)

Steinhauser, Martin O.; Schindler, Tanja

2017-01-01

We report on the results of particle-based, coarse-grained molecular dynamics simulations of amphiphilic lipid molecules in aqueous environment where the membrane structures at equilibrium are subsequently exposed to strong shock waves, and their damage is analyzed. The lipid molecules self-assemble from unbiased random initial configurations to form stable bilayer membranes, including closed vesicles. During self-assembly of lipid molecules, we observe several stages of clustering, starting with many small clusters of lipids, gradually merging together to finally form one single bilayer membrane. We find that the clustering of lipids sensitively depends on the hydrophobic interaction h_c of the lipid tails in our model and on temperature T of the system. The self-assembled bilayer membranes are quantitatively analyzed at equilibrium with respect to their degree of order and their local structure. We also show that—by analyzing the membrane fluctuations and using a linearized theory— we obtain area compression moduli K_A and bending stiffnesses κ _B for our bilayer membranes which are within the experimental range of in vivo and in vitro measurements of biological membranes. We also discuss the density profile and the pair correlation function of our model membranes at equilibrium which has not been done in previous studies of particle-based membrane models. Furthermore, we present a detailed phase diagram of our lipid model that exhibits a sol-gel transition between quasi-solid and fluid domains, and domains where no self-assembly of lipids occurs. In addition, we present in the phase diagram the conditions for temperature T and hydrophobicity h_c of the lipid tails of our model to form closed vesicles. The stable bilayer membranes obtained at equilibrium are then subjected to strong shock waves in a shock tube setup, and we investigate the damage in the membranes due to their interaction with shock waves. Here, we find a transition from self-repairing membranes (reducing their damage after impact) and permanent (irreversible) damage, depending on the shock front speed. The here presented idea of using coarse-grained (CG) particle models for soft matter systems in combination with the investigation of shock-wave effects in these systems is a quite new approach.

Evaluating Force Fields for the Computational Prediction of Ionized Arginine and Lysine Side-Chains Partitioning into Lipid Bilayers and Octanol.

PubMed

Sun, Delin; Forsman, Jan; Woodward, Clifford E

2015-04-14

Abundant peptides and proteins containing arginine (Arg) and lysine (Lys) amino acids can apparently permeate cell membranes with ease. However, the mechanisms by which these peptides and proteins succeed in traversing the free energy barrier imposed by cell membranes remain largely unestablished. Precise thermodynamic studies (both theoretical and experimental) on the interactions of Arg and Lys residues with model lipid bilayers can provide valuable clues to the efficacy of these cationic peptides and proteins. We have carried out molecular dynamics simulations to calculate the interactions of ionized Arg and Lys side-chains with the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayer for 10 widely used lipid/protein force fields: CHARMM36/CHARMM36, SLIPID/AMBER99SB-ILDN, OPLS-AA/OPLS-AA, Berger/OPLS-AA, Berger/GROMOS87, Berger/GROMOS53A6, GROMOS53A6/GROMOS53A6, nonpolarizable MARTINI, polarizable MARTINI, and BMW MARTINI. We performed umbrella sampling simulations to obtain the potential of mean force for Arg and Lys side-chains partitioning from water to the bilayer interior. We found significant differences between the force fields, both for the interactions between side-chains and bilayer surface, as well as the free energy cost for placing the side-chain at the center of the bilayer. These simulation results were compared with the Wimley-White interfacial scale. We also calculated the free energy cost for transferring ionized Arg and Lys side-chains from water to both dry and wet octanol. Our simulations reveal rapid diffusion of water molecules into octanol whereby the equilibrium mole fraction of water in the wet octanol phase was ∼25%. Surprisingly, our free energy calculations found that the high water content in wet octanol lowered the water-to-octanol partitioning free energies for cationic residues by only 0.6 to 0.7 kcal/mol.

Using Micropatterned Lipid Bilayer Arrays to Measure the Effect of Membrane Composition on Merocyanine 540 Binding

PubMed Central

Smith, Kathryn A.; Conboy, John C.

2011-01-01

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 bindingin DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner. PMID:21376014

Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.

PubMed Central

Wong, J Y; Park, C K; Seitz, M; Israelachvili, J

1999-01-01

We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions. PMID:10465756

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

PubMed

Rues, Ralf-Bernhardt; Gräwe, Alexander; Henrich, Erik; Bernhard, Frank

2017-01-01

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant.

PubMed

Asin-Cayuela, Jordi; Manas, Abdul-Rahman B; James, Andrew M; Smith, Robin A J; Murphy, Michael P

2004-07-30

The mitochondria-targeted antioxidant MitoQ comprises a ubiquinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation. This cation drives the membrane potential-dependent accumulation of MitoQ into mitochondria, enabling the ubiquinol antioxidant to prevent mitochondrial oxidative damage far more effectively than untargeted antioxidants. We sought to fine-tune the hydrophobicity of MitoQ so as to control the extent of its membrane binding and penetration into the phospholipid bilayer, and thereby regulate its partitioning between the membrane and aqueous phases within mitochondria and cells. To do this, MitoQ variants with 3, 5, 10 and 15 carbon aliphatic chains were synthesised. These molecules had a wide range of hydrophobicities with octan-1-ol/phosphate buffered saline partition coefficients from 2.8 to 20000. All MitoQ variants were accumulated into mitochondria driven by the membrane potential, but their binding to phospholipid bilayers varied from negligible for MitoQ3 to essentially total for MitoQ15. Despite the span of hydrophobicites, all MitoQ variants were effective antioxidants. Therefore, it is possible to fine-tune the degree of membrane association of MitoQ and other mitochondria targeted compounds, without losing antioxidant efficacy. This indicates how the uptake and distribution of mitochondria-targeted compounds within mitochondria and cells can be controlled, thereby facilitating investigations of mitochondrial oxidative damage.

Beyond apoptosis: the mechanism and function of phosphatidylserine asymmetry in the membrane of activating mast cells.

PubMed

Rysavy, Noel M; Shimoda, Lori M N; Dixon, Alyssa M; Speck, Mark; Stokes, Alexander J; Turner, Helen; Umemoto, Eric Y

2014-01-01

Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation.

Surface dynamics of aerolysin on the plasma membrane of living cells.

PubMed

Abrami, L; Fivaz, M; van der Goot, F G

2000-10-01

Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

Bilayer Poly(Lactic-co-glycolic acid)/Nano-Hydroxyapatite Membrane with Barrier Function and Osteogenesis Promotion for Guided Bone Regeneration

PubMed Central

Fu, Li; Wang, Zhanfeng; Dong, Shujun; Cai, Yan; Ni, Yuxin; Zhang, Tianshou; Wang, Lin; Zhou, Yanmin

2017-01-01

Guided bone regeneration (GBR) is one such treatment that reconstructs neo-bone tissue by using a barrier membrane to prevent the invasion of soft tissue and to create a space for guiding new bone growth into the bone defect. Herein, we report a novel functionally graded bilayer membrane (FGBM) for GBR application. To fabricate the novel membrane, the composites of poly(lactic-co-glycolic acid) and nano-hydroxyapatite were prepared by phase inversion for the dense layer and by electrospinning for another porous layer, and their corresponding properties were evaluated including surface morphology, mechanics, degradability, cell barrier function, and in vitro osteogenic bioactivity. The results showed that PLGA with 5% nHA in dense layer could meet the requirement of mechanical strength and have excellent barrier function even on condition of post-degradation. Furthermore, PLGA with 30% nHA in porous layer could achieve the good physical and chemical properties. In addition, 30% nHA incorporation would enhance the in vitro mineralization, and have superior capabilities of cell adhesion, proliferation and differentiation compared to other groups. Therefore, the designed FGBM could potentially serve as a barrier for preferential tissue ingrowth and achieve a desirable therapeutic result for bone tissue regeneration. PMID:28772618

Apatite nanoparticles strongly improve red blood cell cryopreservation by mediating trehalose delivery via enhanced membrane permeation.

PubMed

Stefanic, Martin; Ward, Kevin; Tawfik, Harvey; Seemann, Ralf; Baulin, Vladimir; Guo, Yachong; Fleury, Jean-Baptiste; Drouet, Christophe

2017-09-01

Cryopreservation of red blood cells (RBC) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. Currently, in a clinical setting, glycerol is used as cryoprotectant against freezing damage. After thawing and before transfusion, glycerol must however be removed to avoid intravascular hemolysis, via a complex and time-consuming deglycerolization process which requires specialized equipment. Improved cryopreservation methods using non-toxic agents are required to increase biocompatibility and decrease processing time. Biocompatible cryoprotectants (e.g. trehalose) were proposed, but their low permeation through RBC membranes limits their cryoprotection efficacy. Herein, we report for the first time a glycerol-free cryopreservation approach, using colloidal bioinspired apatite nanoparticles (NP) as bioactive promoters of RBC cryopreservation mediated by trehalose. Addition of apatite NP in the medium tremendously increases RBC cryosurvival, up to 91% (42% improvement compared to a control without NP) which is comparable to FDA-approved cryoprotection protocol employing glycerol. NP concentration and incubation conditions strongly modulate the NP bioactivity. Complementary experimental and computational analyses of the interaction between apatite NP and model lipid bilayers revealed complex events occurring at the NP-bilayer interface. Apatite NP do not cross the bilayer but momentarily modulate its physical status. These changes affect the membrane behavior, and promote the permeation of trehalose and a model fluorescent molecule (FITC). This approach is a new alternative to using toxic glycerol for cells cryopreservation, and the identification of this enhancing no-pore permeation mechanism of apatite NP appears as an original delivery pathway for cryoprotectant agents and beyond. Copyright © 2017 Elsevier Ltd. All rights reserved.

Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces

PubMed Central

Atanasov, Vladimir; Knorr, Nikolaus; Duran, Randolph S.; Ingebrandt, Sven; Offenhäusser, Andreas; Knoll, Wolfgang; Köper, Ingo

2005-01-01

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin. PMID:16127170

Interaction of partially denatured insulin with a DSPC floating lipid bilayer.

PubMed

Dennison, A J C; Jones, R A L; Staniforth, R A; Parnell, A J

2016-01-21

The carefully controlled permeability of cellular membranes to biological molecules is key to life. In degenerative diseases associated with protein misfolding and aggregation, protein molecules or their aggregates are believed to permeate these barriers and threaten membrane integrity. We used neutron reflectivity to study the interaction of insulin, a model amyloidogenic protein, with a DSPC floating lipid bilayer. Structural changes consistent with protein partitioning to the membrane interior and adsorption to a gel phase model lipid bilayer were observed under conditions where the native fold of the protein is significantly destabilised. We propose that the perturbation of the membrane by misfolded proteins involves long term occupation of the membrane by these proteins, rather than transient perforation events.

The OpenPicoAmp: an open-source planar lipid bilayer amplifier for hands-on learning of neuroscience.

PubMed

Shlyonsky, Vadim; Dupuis, Freddy; Gall, David

2014-01-01

Understanding the electrical biophysical properties of the cell membrane can be difficult for neuroscience students as it relies solely on lectures of theoretical models without practical hands on experiments. To address this issue, we developed an open-source lipid bilayer amplifier, the OpenPicoAmp, which is appropriate for use in introductory courses in biophysics or neurosciences at the undergraduate level, dealing with the electrical properties of the cell membrane. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. We provide a complete documentation allowing to build the amplifier and the experimental chamber. The students hand-out giving step-by step instructions to perform a recording is also included. Our experimental setup can be used in basic experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionic channels like gramicidin A dimers. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics.

The OpenPicoAmp: An Open-Source Planar Lipid Bilayer Amplifier for Hands-On Learning of Neuroscience

PubMed Central

Shlyonsky, Vadim; Dupuis, Freddy; Gall, David

2014-01-01

Understanding the electrical biophysical properties of the cell membrane can be difficult for neuroscience students as it relies solely on lectures of theoretical models without practical hands on experiments. To address this issue, we developed an open-source lipid bilayer amplifier, the OpenPicoAmp, which is appropriate for use in introductory courses in biophysics or neurosciences at the undergraduate level, dealing with the electrical properties of the cell membrane. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. We provide a complete documentation allowing to build the amplifier and the experimental chamber. The students hand-out giving step-by step instructions to perform a recording is also included. Our experimental setup can be used in basic experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionic channels like gramicidin A dimers. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics. PMID:25251830

Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites

PubMed Central

1996-01-01

The protein ankyrin links integral membrane proteins to the spectrin- based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol- linked form of neuroglian failed to recruit ankyrin to sites of cell- cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane. PMID:8636238

Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites.

PubMed

Dubreuil, R R; MacVicar, G; Dissanayake, S; Liu, C; Homer, D; Hortsch, M

1996-05-01

The protein ankyrin links integral membrane proteins to the spectrin-based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol-linked form of neuroglian failed to recruit ankyrin to sites of cell-cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane.

The plasma membrane as a capacitor for energy and metabolism.

PubMed

Ray, Supriyo; Kassan, Adam; Busija, Anna R; Rangamani, Padmini; Patel, Hemal H

2016-02-01

When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as "capacitors for energy and metabolism." Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell.

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

PubMed

Holst, Mikkel Roland; Vidal-Quadras, Maite; Larsson, Elin; Song, Jie; Hubert, Madlen; Blomberg, Jeanette; Lundborg, Magnus; Landström, Maréne; Lundmark, Richard

2017-08-22

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

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

PubMed

Huang, Kun; García, Angel E

2014-10-14

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.

Characterization of femtosecond-laser pulse induced cell membrane nanosurgical attachment.

PubMed

Katchinskiy, Nir; Godbout, Roseline; Elezzabi, Abdulhakem Y

2016-07-01

This article provides insight into the mechanism of femtosecond laser nanosurgical attachment of cells. We have demonstrated that during the attachment of two retinoblastoma cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength, the phospholipid molecules of both cells hemifuse and form one shared phospholipid bilayer, at the attachment location. In order to verify the hypothesis that hemifusion takes place, transmission electron microscope images of the cell membranes of retinoblastoma cells were taken. It is shown that at the attachment interface, the two cell membranes coalesce and form one single membrane shared by both cells. Thus, further evidence is provided to support the hypothesis that laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane, which resulted in cross-linking of the phospholipid molecules in each membrane. This process of hemifusion occurs throughout the entire penetration depth of the femtosecond laser pulse train. Thus, the attachment between the cells takes place across a large surface area, which affirms our findings of strong physical attachment between the cells. The femtosecond laser pulse hemifusion technique can potentially provide a platform for precise molecular manipulation of cellular membranes. Manipulation of the cellular membrane is an important procedure that could aid in studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

One-dimensional potential of mean force underestimates activation barrier for transport across flexible lipid membranes

NASA Astrophysics Data System (ADS)

Kopelevich, Dmitry I.

2013-10-01

Transport of a fullerene-like nanoparticle across a lipid bilayer is investigated by coarse-grained molecular dynamics (MD) simulations. Potentials of mean force (PMF) acting on the nanoparticle in a flexible bilayer suspended in water and a bilayer restrained to a flat surface are computed by constrained MD simulations. The rate of the nanoparticle transport into the bilayer interior is predicted using one-dimensional Langevin models based on these PMFs. The predictions are compared with the transport rates obtained from a series of direct (unconstrained) MD simulations of the solute transport into the flexible bilayer. It is observed that the PMF acting on the solute in the flexible membrane underestimates the transport rate by more than an order of magnitude while the PMF acting on the solute in the restrained membrane yields an accurate estimate of the activation energy for transport into the flexible membrane. This paradox is explained by a coexistence of metastable membrane configurations for a range of the solute positions inside and near the flexible membrane. This leads to a significant reduction of the contribution of the transition state to the mean force acting on the solute. Restraining the membrane shape ensures that there is only one stable membrane configuration corresponding to each solute position and thus the transition state is adequately represented in the PMF. This mechanism is quite general and thus this phenomenon is expected to occur in a wide range of interfacial systems. A simple model for the free energy landscape of the coupled solute-membrane system is proposed and validated. This model explicitly accounts for effects of the membrane deformations on the solute transport and yields an accurate prediction of the activation energy for the solute transport.

RNA and DNA interactions with zwitterionic and charged lipid membranes - a DSC and QCM-D study.

PubMed

Michanek, Agnes; Kristen, Nora; Höök, Fredrik; Nylander, Tommy; Sparr, Emma

2010-04-01

The aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na(+)) or divalent (Ca(2+)) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC-DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC-DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA-phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine. Copyright 2010 Elsevier B.V. All rights reserved.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

PubMed

Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

2013-04-01

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013 Elsevier B.V. All rights reserved.

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine Fluorophore into a Phospholipid Bilayer: Complementary Use of Fluorescence Quenching Studies and Molecular Dynamics Simulations

PubMed Central

Kyrychenko, Alexander; Sevriukov, Igor Yu.; Syzova, Zoya A.; Ladokhin, Alexey S.; Doroshenko, Andrey O.

2014-01-01

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from -6.5 to -7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP. PMID:21211898

Transport methods for probing the barrier domain of lipid bilayer membranes.

PubMed Central

Xiang, T X; Chen, X; Anderson, B D

1992-01-01

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent. PMID:1420875

Phospholipid Diffusion Coefficients of Cushioned Model Membranes determined via Z-Scan Fluorescence Correlation Spectroscopy

PubMed Central

Sterling, Sarah M.; Allgeyer, Edward S.; Fick, Jörg; Prudovsky, Igor; Mason, Michael D.; Neivandt, David J.

2013-01-01

Model cellular membranes enable the study of biological processes in a controlled environment and reduce the traditional challenges associated with live or fixed cell studies. However, model membrane systems based on the air/water or oil/solution interface do not allow for incorporation of transmembrane proteins, or for the study of protein transport mechanisms. Conversely, a phospholipid bilayer deposited via the Langmuir-Blodgett/Langmuir Schaefer method on a hydrogel layer is potentially an effective mimic of the cross-section of a biological membrane, and facilitates both protein incorporation and transport studies. Prior to application, however, such membranes must be fully characterized, particularly with respect to the phospholipid bilayer phase transition temperature. Here we present a detailed characterization of the phase transition temperature of the inner and outer leaflets of a chitosan supported model membrane system. Specifically, the lateral diffusion coefficient of each individual leaflet has been determined as a function of temperature. Measurements were performed utilizing z-scan fluorescence correlation spectroscopy (FCS), a technique that yields calibration-free diffusion information. Analysis via the method of Wawrezinieck and coworkers, revealed that phospholipid diffusion changes from raft-like to free diffusion as the temperature is increased; an insight into the dynamic behavior of hydrogel supported membranes not previously reported. PMID:23705855

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature.

PubMed

Cheney, Philip P; Weisgerber, Alan W; Feuerbach, Alec M; Knowles, Michelle K

2017-03-15

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl- sn -glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

NASA Astrophysics Data System (ADS)

Li, Jing; Liu, Xiuhua; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Cao, Dapeng

2018-05-01

GPI-Anchored proteins (GPI-APs) can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

Graphene can wreak havoc with cell membranes.

PubMed

Dallavalle, Marco; Calvaresi, Matteo; Bottoni, Andrea; Melle-Franco, Manuel; Zerbetto, Francesco

2015-02-25

Molecular dynamics--coarse grained to the level of hydrophobic and hydrophilic interactions--shows that small hydrophobic graphene sheets pierce through the phospholipid membrane and navigate the double layer, intermediate size sheets pierce the membrane only if a suitable geometric orientation is met, and larger sheets lie mainly flat on the top of the bilayer where they wreak havoc with the membrane and create a patch of upturned phospholipids. The effect arises in order to maximize the interaction between hydrophobic moieties and is quantitatively explained in terms of flip-flops by the analysis of the simulations. Possible severe biological consequences are discussed.

Study of the Interaction of the HIV-1 Fusion Peptide with Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Heller, William; Rai, Durgesh

HIV-1 undergoes fusion with the cell membrane through interactions between its coat proteins and the target cell. Visualization of fusion with sufficient detail to determine the molecular mechanism remains elusive. Here, the interaction between a synthetic variant of the HIV-1 gp41 fusion peptide with vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was studied. The peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet that is accompanied by a transition in the structure of the lipid bilayer vesicle. The peptide changes the distribution of lipids between the vesicle leaflets. Further, it creates two regions having different thicknesses. The results shed new light on how the peptide modifies the membrane structure to favor fusion. A portion of this research was sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U. S. Department of Energy. Research at Oak Ridge National Laboratory's Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U. S. Department of Energy.

Free-Standing Bilayered Nanoparticle Superlattice Nanosheets with Asymmetric Ionic Transport Behaviors.

PubMed

Rao, Siyuan; Si, Kae Jye; Yap, Lim Wei; Xiang, Yan; Cheng, Wenlong

2015-11-24

Natural cell membranes can directionally and selectively regulate the ion transport, which is critical for the functioning of living cells. Here, we report on the fabrication of an artificial membrane based on an asymmetric nanoparticle superlattice bilayered nanosheet, which exhibits similar ion transport characteristics. The superlattice nanosheets were fabricated via a drying-mediated self-assembly of polystyrene-capped gold nanoparticles at the liquid-air interface. By adopting a layer-by-layer assembly process, an asymmetric nanomembrane could be obtained consisting of two nanosheets with different nanoparticle size. The resulting nanomembranes exhibit an asymmetric ion transport behavior, and diode-like current-voltage curves were observed. The asymmetric ion transport is attributed to the cone-like nanochannels formed within the membranes, upon which a simulation map was established to illustrate the relationship between the channel structure and the ionic selectivity, in consistency with our experimental results. Our superlattice nanosheet-based design presents a promising strategy for the fabrication of next-generation smart nanomembranes for rationally and selectively regulating the ion transport even at a large ion flux, with potential applications in a wide range of fields, including biosensor devices, energy conversion, biophotonics, and bioelectronics.

MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli*

PubMed Central

Renner, Lars D.; Weibel, Douglas B.

2012-01-01

The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (Kd) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The Kd for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (kon). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential. PMID:23012351

Mechanism of Azalomycin F5a against Methicillin-Resistant Staphylococcus aureus

PubMed Central

Xu, Li; Xu, Xuejie; Wang, Yimin; Liu, Erxiao

2018-01-01

To investigate the mechanism of azalomycin F5a against methicillin-resistant Staphylococcus aureus (MRSA), the conductivity of MRSA suspension and the adenylate kinase activity of MRSA culture were determined with the intervention of azalomycin F5a, which were significantly increased compared to those of blank controls. This inferred that azalomycin F5a could lead to the leakage of cellular substances possibly by increasing permeability to kill MRSA. As phospholipid bilayer was mainly responsible for cell-membrane permeability, the interaction between azalomycin F5a and cell-membrane lipids was further researched by determining the anti-MRSA activities of azalomycin F5a combined with cell-membrane lipids extracted from test MRSA or with 1,2-dipalmitoyl-sn-glycero-3-phospho-glycerol (DPPG) for possible molecular targets lying in MRSA cell-membrane. The results indicated that the anti-MRSA activity of azalomycin F5a remarkably decreased when it combined with membrane lipids or DPPG. This indicated that cell-membrane lipids especially DPPG might be important targets of azalomycin F5a against MRSA. PMID:29607325

pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.

PubMed Central

Kamp, F; Hamilton, J A

1992-01-01

A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from approximately 50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 < or = 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia. Images PMID:1454821

Membrane solubilisation and reconstitution by octylglucoside: comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry.

PubMed

Krylova, Oxana O; Jahnke, Nadin; Keller, Sandro

2010-08-01

We have studied the solubilisation and reconstitution of lipid membranes composed of either synthetic phosphatidylcholine or Escherichia. coli polar lipid extract by the non-ionic detergent octylglucoside. For both lipid systems, composition-dependent transformations of unilamellar vesicles into micelles or vice versa were followed by high-sensitivity isothermal titration calorimetry. Data obtained over a range of detergent and lipid concentrations could be rationalised in terms of a three-stage phase separation model involving bilayer, bilayer/micelle coexistence, and micellar ranges, yielding the detergent/lipid phase diagrams and the bilayer-to-micelle partition coefficients of both detergent and lipid. The most notable difference between the lipids investigated was a substantial widening of the bilayer/micelle coexistence range for E. coli lipid, which was due to an increased preference of the detergent and a decreased affinity of the lipid for the micellar phase as compared with the bilayer phase. These effects on the bilayer-to-micelle partition coefficients could be explained by the high proportion in E. coli membranes of lipids possessing negative spontaneous curvature, which hampers both their transfer into strongly curved micellar structures as well as the insertion of detergent into condensed bilayers.

Cyclic and Linear Monoterpenes in Phospholipid Membranes: Phase Behavior, Bilayer Structure, and Molecular Dynamics.

PubMed

Pham, Quoc Dat; Topgaard, Daniel; Sparr, Emma

2015-10-13

Monoterpenes are abundant in essential oils extracted from plants. These relatively small and hydrophobic molecules have shown important biological functions, including antimicrobial activity and membrane penetration enhancement. The interaction between the monoterpenes and lipid bilayers is considered important to the understanding of the biological functions of monoterpenes. In this study, we investigated the effect of cyclic and linear monoterpenes on the structure and dynamics of lipids in model membranes. We have studied the ternary system 1,2-dimyristoyl-sn-glycero-3-phosphocholine-monoterpene-water as a model with a focus on dehydrated conditions. By combining complementary techniques, including differential scanning calorimetry, solid-state nuclear magnetic resonance, and small- and wide-angle X-ray scattering, bilayer structure, phase transitions, and lipid molecular dynamics were investigated at different water contents. Monoterpenes cause pronounced melting point depression and phase segregation in lipid bilayers, and the extent of these effects depends on the hydration conditions. The addition of a small amount of thymol to the fluid bilayer (volume fraction of 0.03 in the bilayer) leads to an increased order in the acyl chain close to the bilayer interface. The findings are discussed in relation to biological systems and lipid formulations.

Poly(aniline) nanowires in sol-gel coated ITO: A pH-responsive substrate for planar supported lipid bilayers

PubMed Central

Ge, Chenhao; Orosz, Kristina S.; Armstrong, Neal R.; Saavedra, S. Scott

2011-01-01

Facilitated ion transport across an artificial lipid bilayer coupled to a solid substrate is a function common to several types of bioelectronic devices based on supported membranes, including biomimetic fuel cells and ion channel biosensors. Described here is fabrication of a pH-sensitive transducer composed of a porous sol-gel layer derivatized with poly(aniline) (PANI) nanowires grown from an underlying planar indium-tin oxide (ITO) electrode. The upper sol-gel surface is hydrophilic, smooth, and compatible with deposition of a planar supported lipid bilayer (PSLB) formed via vesicle fusion. Conducting tip AFM was used to show that the PANI wires are connected to the ITO, which convert this electrode into a potentiometric pH sensor. The response to changes in the pH of the buffer contacting the PANI nanowire/sol-gel/ITO electrode is blocked by the very low ion permeability of the overlying, fluid PSLB. The feasibility of using this assembly to monitor facilitated proton transport across the PSLB was demonstrated by doping the membrane with lipophilic ionophores that respond to a transmembrane pH gradient, which produced an apparent proton permeability several orders of magnitude greater than values measured for undoped lipid bilayers. PMID:21707069

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes.

PubMed

Lopez, Victor; Cauvi, David M; Arispe, Nelson; De Maio, Antonio

2016-07-01

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

NASA Astrophysics Data System (ADS)

Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

2010-05-01

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

Modulation of physiological and pathological activities of lysozyme by biological membranes.

PubMed

Trusova, Valeriya

2012-09-01

The molecular details of interactions between lipid membranes and lysozyme (Lz), a small polycationic protein with a wide range of biological activities, have long been the focus of numerous studies. The biological consequences of this process are considered to embrace at least two aspects: i) correlation between antimicrobial and membranotropic properties of this protein, and ii) lipid-mediated Lz amyloidogenesis. The mechanisms underlying the lipid-assisted protein fibrillogenesis and membrane disruption exerted by Lz in bacterial cells are believed to be similar. The present investigation was undertaken to gain further insight into Lz-lipid interactions and explore the routes by which Lz exerts its antimicrobial and amyloidogenic actions. Binding and Förster resonance energy transfer studies revealed that upon increasing the content of anionic lipids in lipid vesicles, Lz forms aggregates in a membrane environment. Total internal reflection fluorescence microscopy and pyrene excimerization reaction were employed to study the effect of Lz on the structural and dynamic properties of lipid bilayers. It was found that Lz induces lipid demixing and reduction of bilayer free volume, the magnitude of this effect being much more pronounced for oligomeric protein.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Jeremy C.; Cheng, Xiaolin; Nickels, Jonathan D.

Understanding of cell membrane organization has evolved significantly from the classic fluid mosaic model. It is now recognized that biological membranes are highly organized structures, with differences in lipid compositions between inner and outer leaflets and in lateral structures within the bilayer plane, known as lipid rafts. These organizing principles are important for protein localization and function as well as cellular signaling. However, the mechanisms and biophysical basis of lipid raft formation, structure, dynamics and function are not clearly understood. One key question, which we focus on in this review, is how lateral organization and leaflet compositional asymmetry are coupled.more » Detailed information elucidating this question has been sparse because of the small size and transient nature of rafts and the experimental challenges in constructing asymmetric bilayers. Resolving this mystery will require advances in both experimentation and modeling. We discuss here the preparation of model systems along with experimental and computational approaches that have been applied in efforts to address this key question in membrane biology. Furthermore, we seek to place recent and future advances in experimental and computational techniques in context, providing insight into in-plane and transverse organization of biological membranes.« less

Probing molecular interactions of poly(styrene-co-maleic acid) with lipid matrix models to interpret the therapeutic potential of the co-polymer.

PubMed

Banerjee, Shubhadeep; Pal, Tapan K; Guha, Sujoy K

2012-03-01

To understand and maximize the therapeutic potential of poly(styrene-co-maleic acid) (SMA), a synthetic, pharmacologically-active co-polymer, its effect on conformation, phase behavior and stability of lipid matrix models of cell membranes were investigated. The modes of interaction between SMA and lipid molecules were also studied. While, attenuated total reflection-Fourier-transform infrared (ATR-FTIR) and static (31)P nuclear magnetic resonance (NMR) experiments detected SMA-induced conformational changes in the headgroup region, differential scanning calorimetry (DSC) studies revealed thermotropic phase behavior changes of the membranes. (1)H NMR results indicated weak immobilization of SMA within the bilayers. Molecular interpretation of the results indicated the role of hydrogen-bond formation and hydrophobic forces between SMA and zwitterionic phospholipid bilayers. The extent of membrane fluidization and generation of isotropic phases were affected by the surface charge of the liposomes, and hence suggested the role of electrostatic interactions between SMA and charged lipid headgroups. SMA was thus found to directly affect the structural integrity of model membranes. Copyright © 2011 Elsevier B.V. All rights reserved.

Membrane Perturbation Induced by Interfacially Adsorbed Peptides

PubMed Central

Zemel, Assaf; Ben-Shaul, Avinoam; May, Sylvio

2004-01-01

The structural and energetic characteristics of the interaction between interfacially adsorbed (partially inserted) α-helical, amphipathic peptides and the lipid bilayer substrate are studied using a molecular level theory of lipid chain packing in membranes. The peptides are modeled as “amphipathic cylinders” characterized by a well-defined polar angle. Assuming two-dimensional nematic order of the adsorbed peptides, the membrane perturbation free energy is evaluated using a cell-like model; the peptide axes are parallel to the membrane plane. The elastic and interfacial contributions to the perturbation free energy of the “peptide-dressed” membrane are evaluated as a function of: the peptide penetration depth into the bilayer's hydrophobic core, the membrane thickness, the polar angle, and the lipid/peptide ratio. The structural properties calculated include the shape and extent of the distorted (stretched and bent) lipid chains surrounding the adsorbed peptide, and their orientational (C-H) bond order parameter profiles. The changes in bond order parameters attendant upon peptide adsorption are in good agreement with magnetic resonance measurements. Also consistent with experiment, our model predicts that peptide adsorption results in membrane thinning. Our calculations reveal pronounced, membrane-mediated, attractive interactions between the adsorbed peptides, suggesting a possible mechanism for lateral aggregation of membrane-bound peptides. As a special case of interest, we have also investigated completely hydrophobic peptides, for which we find a strong energetic preference for the transmembrane (inserted) orientation over the horizontal (adsorbed) orientation. PMID:15189858

Jumping acoustic bubbles on lipid bilayers.

PubMed

Der Loughian, Christelle; Muleki Seya, Pauline; Pirat, Christophe; Inserra, Claude; Béra, Jean-Christophe; Rieu, Jean-Paul

2015-05-07

In the context of sonoporation, we use supported lipid bilayers as a model for biological membranes and investigate the interactions between the bilayer and microbubbles induced by ultrasound. Among the various types of damage caused by bubbles on the surface, our experiments exhibit a singular dynamic interaction process where bubbles are jumping on the bilayer, forming a necklace pattern of alteration on the membrane. This phenomenon was explored with different time and space resolutions and, based on our observations, we propose a model for a microbubble subjected to the combined action of van der Waals, acoustic and hydrodynamic forces. Describing the repeated jumps of the bubble, this model explains the lipid exchanges between the bubble and bilayer.

Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

PubMed

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

2012-11-01

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun

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

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

Molecular binding of catechins to biomembranes:Relationship to biolgical activity

USDA-ARS?s Scientific Manuscript database

Molecular dynamics simulations were used to study the interactions of four catechins commonly found in tea with lipid bilayers of cell membranes. Experimental studies in the literature have shown that catechins are linked to beneficial health effects, specifically as they are related to interaction...

High-Throughput Simulations Reveal Membrane-Mediated Effects of Alcohols on MscL Gating

PubMed Central

2017-01-01

The mechanosensitive channels of large conductance (MscL) are bacterial membrane proteins that serve as last resort emergency release valves in case of severe osmotic downshock. Sensing bilayer tension, MscL channels are sensitive to changes in the bilayer environment and are, therefore, an ideal test case for exploring membrane protein coupling. Here, we use high-throughput coarse-grained molecular dynamics simulations to characterize MscL gating kinetics in different bilayer environments under the influence of alcohols. We performed over five hundred simulations to obtain sufficient statistics to reveal the subtle effects of changes in the membrane environment on MscL gating. MscL opening times were found to increase with the addition of the straight-chain alcohols ethanol, octanol, and to some extent dodecanol but not with hexadecanol. Increasing concentration of octanol increased the impeding effect, but only up to 10–20 mol %. Our in silico predictions were experimentally confirmed using reconstituted MscL in a liposomal fluorescent efflux assay. Our combined data reveal that the effect of alcohols on MscL gating arises not through specific binding sites but through a combination of the alcohol-induced changes to a number of bilayer properties and their alteration of the MscL–bilayer interface. Our work provides a key example of how extensive molecular simulations can be used to predict the functional modification of membrane proteins by subtle changes in their bilayer environment. PMID:28122455

High-throughput simulations reveal membrane-mediated effects of alcohols on MscL gating

DOE PAGES

Melo, Manuel N.; Arnarez, Clement; Sikkema, Hendrik; ...

2017-01-26

The mechanosensitive channels of large conductance (MscL) are bacterial membrane proteins that serve as last resort emergency release valves in case of severe osmotic downshock. Sensing bilayer tension, MscL channels are sensitive to changes in the bilayer environment and are, therefore, an ideal test case for exploring membrane protein coupling. Here, we use high-throughput coarse-grained molecular dynamics simulations to characterize MscL gating kinetics in different bilayer environments under the influence of alcohols. We performed over five hundred simulations to obtain sufficient statistics to reveal the subtle effects of changes in the membrane environment on MscL gating. MscL opening times weremore » found to increase with the addition of the straight-chain alcohols ethanol, octanol, and to some extent dodecanol but not with hexadecanol. Increasing concentration of octanol increased the impeding effect, but only up to 10–20 mol %. Our in silico predictions were experimentally confirmed using reconstituted MscL in a liposomal fluorescent efflux assay. Our combined data reveal that the effect of alcohols on MscL gating arises not through specific binding sites but through a combination of the alcohol-induced changes to a number of bilayer properties and their alteration of the MscL–bilayer interface. Finally, our work provides a key example of how extensive molecular simulations can be used to predict the functional modification of membrane proteins by subtle changes in their bilayer environment.« less

Sensing small molecule interactions with lipid membranes by local pH modulation.

PubMed

Huang, Da; Zhao, Tao; Xu, Wei; Yang, Tinglu; Cremer, Paul S

2013-11-05

Herein, we utilized a label-free sensing platform based on pH modulation to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic, and planar supported lipid bilayers (SLBs). The SLBs were patterned inside a flow cell, allowing for various concentrations of tetracaine to be introduced over the surface in a buffer solution. Studies with membranes containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180 ± 47 μm for this small molecule-membrane interaction. Adding cholesterol to the SLBs decreased the affinity between tetracaine and the bilayers, while this interaction tightened when POPE (1-hexadecanoyl-2-(9-Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) was added. Studies were also conducted with three negatively charged membrane lipids, POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)), and ganglioside GM1. All three measurements gave rise to a similar tightening of the apparent Kd value compared with pure POPC membranes. The lack of chemical specificity with the identity of the negatively charged lipid indicated that the tightening was largely electrostatic. Through a direct comparison with ITC measurements, it was found that the pH modulation sensor platform offers a facile, inexpensive, highly sensitive, and rapid method for the detection of interactions between putative drug candidates and lipid bilayers. As such, this technique may potentially be exploited as a screen for drug development and analysis.

Transmembrane proteins in the Protein Data Bank: identification and classification.

PubMed

Tusnády, Gábor E; Dosztányi, Zsuzsanna; Simon, István

2004-11-22

Integral membrane proteins play important roles in living cells. Although these proteins are estimated to constitute 25% of proteins at a genomic scale, the Protein Data Bank (PDB) contains only a few hundred membrane proteins due to the difficulties with experimental techniques. The presence of transmembrane proteins in the structure data bank, however, is quite invisible, as the annotation of these entries is rather poor. Even if a protein is identified as a transmembrane one, the possible location of the lipid bilayer is not indicated in the PDB because these proteins are crystallized without their natural lipid bilayer, and currently no method is publicly available to detect the possible membrane plane using the atomic coordinates of membrane proteins. Here, we present a new geometrical approach to distinguish between transmembrane and globular proteins using structural information only and to locate the most likely position of the lipid bilayer. An automated algorithm (TMDET) is given to determine the membrane planes relative to the position of atomic coordinates, together with a discrimination function which is able to separate transmembrane and globular proteins even in cases of low resolution or incomplete structures such as fragments or parts of large multi chain complexes. This method can be used for the proper annotation of protein structures containing transmembrane segments and paves the way to an up-to-date database containing the structure of all known transmembrane proteins and fragments (PDB_TM) which can be automatically updated. The algorithm is equally important for the purpose of constructing databases purely of globular proteins.

Lennard-Jones type pair-potential method for coarse-grained lipid bilayer membrane simulations in LAMMPS

NASA Astrophysics Data System (ADS)

Fu, S.-P.; Peng, Z.; Yuan, H.; Kfoury, R.; Young, Y.-N.

2017-01-01

Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiencies have been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4 ∼ 6 nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane, such as gel-fluid-gas phase transitions of lipids, diffusion, and bending rigidity Yuan et al. (2010). In this work we implement such an interaction potential in LAMMPS to simulate large-scale lipid systems such as a giant unilamellar vesicle (GUV) and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for RBC dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. To demonstrate that the proposed methods do capture the observed dynamics of vesicles and RBCs, we focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with enclosed volume; 2. RBC shape transitions with different enclosed volume. Finally utilizing the parallel computing capability in LAMMPS, we provide some timing results for parallel coarse-grained simulations to illustrate that it is possible to use LAMMPS to simulate large-scale realistic complex biological membranes for more than 1 ms.

Influence of natural organic matter (NOM) coatings on nanoparticle adsorption onto supported lipid bilayers.

PubMed

Bo, Zhang; Avsar, Saziye Yorulmaz; Corliss, Michael K; Chung, Minsub; Cho, Nam-Joon

2017-10-05

As the worldwide usage of nanoparticles in commercial products continues to increase, there is growing concern about the environmental risks that nanoparticles pose to biological systems, including potential damage to cellular membranes. A detailed understanding of how different types of nanoparticles behave in environmentally relevant conditions is imperative for predicting and mitigating potential membrane-associated toxicities. Herein, we investigated the adsorption of two popular nanoparticles (silver and buckminsterfullerene) onto biomimetic supported lipid bilayers of varying membrane charge (positive and negative). The quartz crystal microbalance-dissipation (QCM-D) measurement technique was employed to track the adsorption kinetics. Particular attention was focused on understanding how natural organic matter (NOM) coatings affect nanoparticle-bilayer interactions. Both types of nanoparticles preferentially adsorbed onto the positively charged bilayers, although NOM coatings on the nanoparticle and lipid bilayer surfaces could either inhibit or promote adsorption in certain electrolyte conditions. While past findings showed that NOM coatings inhibit membrane adhesion, our findings demonstrate that the effects of NOM coatings are more nuanced depending on the type of nanoparticle and electrolyte condition. Taken together, the results demonstrate that NOM coatings can modulate the lipid membrane interactions of various nanoparticles, suggesting a possible way to improve the environmental safety of nanoparticles. Copyright © 2017 Elsevier B.V. All rights reserved.

Proton Gradients as a Key Physical Factor in the Evolution of the Forced Transport Mechanism Across the Lipid Membrane.

PubMed

Strbak, Oliver; Kanuchova, Zuzana; Krafcik, Andrej

2016-11-01

A critical phase in the transition from prebiotic chemistry to biological evolution was apparently an asymmetric ion flow across the lipid membrane. Due to imbalance in the ion flow, the early lipid vesicles could selectively take the necessary molecules from the environment, and release the side-products from the vesicle. Natural proton gradients played a definitively crucial role in this process, since they remain the basis of energy transfer in the present-day cells. On the basis of this supposition, and the premise of the early vesicle membrane's impermeability to protons, we have shown that the emergence of the proton gradient in the lipid vesicle could be a key physical factor in the evolution of the forced transport mechanism (pore formation and active transport) across the lipid bilayer. This driven flow of protons across the membrane is the result of the electrochemical proton gradient and osmotic pressures on the integrity of the lipid vesicle. At a critical number of new lipid molecules incorporated into the vesicle, the energies associated with the creation of the proton gradient exceed the bending stiffness of the lipid membrane, and overlap the free energy of the lipid bilayer pore formation.

Molecular dynamics simulations showing 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) membrane mechanoporation damage under different strain paths.

PubMed

Murphy, M A; Mun, Sungkwang; Horstemeyer, M F; Baskes, M I; Bakhtiary, A; LaPlaca, Michelle C; Gwaltney, Steven R; Williams, Lakiesha N; Prabhu, R K

2018-04-09

Continuum finite element material models used for traumatic brain injury lack local injury parameters necessitating nanoscale mechanical injury mechanisms be incorporated. One such mechanism is membrane mechanoporation, which can occur during physical insults and can be devastating to cells, depending on the level of disruption. The current study investigates the strain state dependence of phospholipid bilayer mechanoporation and failure. Using molecular dynamics, a simplified membrane, consisting of 72 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) phospholipids, was subjected to equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial tensile deformations at a von Mises strain rate of 5.45 × 10 8 s -1 , resulting in velocities in the range of 1 to 4.6 m·s -1 . A water bridge forming through both phospholipid bilayer leaflets was used to determine structural failure. The stress magnitude, failure strain, headgroup clustering, and damage responses were found to be strain state-dependent. The strain state order of detrimentality in descending order was equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial. The phospholipid bilayer failed at von Mises strains of .46, .47, .53, .77, and 1.67 during these respective strain path simulations. Additionally, a Membrane Failure Limit Diagram (MFLD) was created using the pore nucleation, growth, and failure strains to demonstrate safe and unsafe membrane deformation regions. This MFLD allowed representative equations to be derived to predict membrane failure from in-plane strains. These results provide the basis to implement a more accurate mechano-physiological internal state variable continuum model that captures lower length scale damage and will aid in developing higher fidelity injury models.

Pt and PtRu catalyst bilayers increase efficiencies for ethanol oxidation in proton exchange membrane electrolysis and fuel cells

NASA Astrophysics Data System (ADS)

Altarawneh, Rakan M.; Pickup, Peter G.

2017-10-01

Polarization curves, product distributions, and reaction stoichiometries have been measured for the oxidation of ethanol at anodes consisting of Pt and PtRu bilayers and a homogeneous mixture of the two catalysts. These anode structures all show synergies between the two catalysts that can be attributed to the oxidation of acetaldehyde produced at the PtRu catalyst by the Pt catalyst. The use of a PtRu layer over a Pt layer produces the strongest effect, with higher currents than a Pt on PtRu bilayer, mixed layer, or either catalyst alone, except for Pt at high potentials. Reaction stoichiometries (average number of electrons transferred per ethanol molecule) were closer to the values for Pt alone for both of the bilayer configurations but much lower for PtRu and mixed anodes. Although Pt alone would provide the highest overall fuel cell efficiency at low power densities, the PtRu on Pt bilayer would provide higher power densities without a significant loss of efficiency. The origin of the synergy between the Pt and PtRu catalysts was elucidated by separation of the total current into the individual components for generation of carbon dioxide and the acetaldehyde and acetic acid byproducts.

Translocation Thermodynamics of Linear and Cyclic Nonaarginine into Model DPPC Bilayer via Coarse-Grained Molecular Dynamics Simulation: Implications of Pore Formation and Nonadditivity

PubMed Central

2015-01-01

Structural mechanisms and underlying thermodynamic determinants of efficient internalization of charged cationic peptides (cell-penetrating peptides, CPPs) such as TAT, polyarginine, and their variants, into cells, cellular constructs, and model membrane/lipid bilayers (large and giant unilamellar or multilamelar vesicles) continue to garner significant attention. Two widely held views on the translocation mechanism center on endocytotic and nonendocytotic (diffusive) processes. Espousing the view of a purely diffusive internalization process (supported by recent experimental evidence, [Säälik, P.; et al. J. Controlled Release2011, 153, 117–125]), we consider the underlying free energetics of the translocation of a nonaarginine peptide (Arg9) into a model DPPC bilayer. In the case of the Arg9 cationic peptide, recent experiments indicate a higher internalization efficiency of the cyclic structure (cyclic Arg9) relative to the linear conformer. Furthermore, recent all-atom resolution molecular dynamics simulations of cyclic Arg9 [Huang, K.; et al. Biophys. J., 2013, 104, 412–420] suggested a critical stabilizing role of water- and lipid-constituted pores that form within the bilayer as the charged Arg9 translocates deep into the bilayer center. Herein, we use umbrella sampling molecular dynamics simulations with coarse-grained Martini lipids, polarizable coarse-grained water, and peptide to explore the dependence of translocation free energetics on peptide structure and conformation via calculation of potentials of mean force along preselected reaction paths allowing and preventing membrane deformations that lead to pore formation. Within the context of the coarse-grained force fields we employ, we observe significant barriers for Arg9 translocation from bulk aqueous solution to bilayer center. Moreover, we do not find free-energy minima in the headgroup–water interfacial region, as observed in simulations using all-atom force fields. The pore-forming paths systematically predict lower free-energy barriers (ca. 90 kJ/mol lower) than the non pore-forming paths, again consistent with all-atom force field simulations. The current force field suggests no preference for the more compact or covalently cyclic structures upon entering the bilayer. Decomposition of the PMF into the system’s components indicates that the dominant stabilizing contribution along the pore-forming path originates from the membrane as both layers of it deformed due to the formation of pore. Furthermore, our analysis revealed that although there is significant entropic stabilization arising from the enhanced configurational entropy exposing more states as the peptide moves through the bilayer, the enthalpic loss (as predicted by the interactions of this coarse-grained model) far outweighs any former stabilization, thus leading to significant barrier to translocation. Finally, we observe reduction in the translocation free-energy barrier for a second Arg9 entering the bilayer in the presence of an initial peptide restrained at the center, again, in qualitative agreement with all-atom force fields. PMID:24506488

Shaping the Flavivirus Replication Complex: It's Curvaceous!

PubMed

Aktepe, Turgut E; Mackenzie, Jason M

2018-06-22

Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus-induced organelles characteristic of infection. This article is protected by copyright. All rights reserved.

Neutron diffraction studies of viral fusion peptides

NASA Astrophysics Data System (ADS)

Bradshaw, Jeremy P.; J. M. Darkes, Malcolm; Katsaras, John; Epand, Richard M.

2000-03-01

Membrane fusion plays a vital role in a large and diverse number of essential biological processes. Despite this fact, the precise molecular events that occur during fusion are still not known. We are currently engaged on a study of membrane fusion as mediated by viral fusion peptides. These peptides are the N-terminal regions of certain viral envelope proteins that mediate the process of fusion between the viral envelope and the membranes of the host cell during the infection process. As part of this study, we have carried out neutron diffraction measurements at the ILL, BeNSC and Chalk River, on a range of viral fusion peptides. The peptides, from simian immunodeficiency virus (SIV), influenza A and feline leukaemia virus (FeLV), were incorporated into stacked phospholipid bilayers. Some of the peptides had been specifically deuterated at key amino acids. Lamellar diffraction data were collected and analysed to yield information on the peptide conformation, location and orientation relative to the bilayer.

Beyond apoptosis: The mechanism and function of phosphatidylserine asymmetry in the membrane of activating mast cells

PubMed Central

Rysavy, Noel M.; Shimoda, Lori M. N.; Dixon, Alyssa M.; Speck, Mark; Stokes, Alexander J.; Turner, Helen; Umemoto, Eric Y.

2014-01-01

Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation. PMID:25759911

Investigation of Dendrimer-Membrane Interactions

NASA Astrophysics Data System (ADS)

Mecke, Almut; Hessler, Jessica; Lee, Inhan; Banaszak Holl, Mark; Orr, Bradford; Patri, Anil K.; Baker, J. R.

2003-03-01

Modified Polyamidoamine (PAMAM) dendrimers show great promise as targeted drug transport agents. Current research efforts point to the possibility of dramatic improvements to conventional chemotherapy by selectively delivering a therapeutic to antigen bearing tumor cells. In order to better understand the uptake mechanism of such devices into cells we are investigating dendrimer-surface adsorption and dendrimer-membrane interactions using atomic force microscopy, light scattering and computer simulations. Model systems consisting of supported DMPC lipid bilayers have shown interesting results suggesting the shape and architecture of nano-devices play an important role for their biologic activity. We are also investigating the effect of targeted drug vehicles on cells in vitro.

Interactions between Ether Phospholipids and Cholesterol as Determined by Scattering and Molecular Dynamics Simulations

PubMed Central

Pan, Jianjun; Cheng, Xiaolin; Heberle, Frederick A.; Mostofian, Barmak; Kučerka, Norbert; Drazba, Paul; Katsaras, John

2012-01-01

Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol’s molecular interactions with ether lipids as determined using a combination of small-angle neutron and X-ray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From the analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data. This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes, cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts, cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup’s phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules. PMID:23199292

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.

Anion channels in the sea urchin sperm plasma membrane.

PubMed

Morales, E; de la Torre, L; Moy, G W; Vacquier, V D; Darszon, A

1993-10-01

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3- > CNS- > Br- > Cl-. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4'-diisothiocyano-2,2'-stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS.

Parvovirus B19 VLP recognizes globoside in supported lipid bilayers.

PubMed

Nasir, Waqas; Nilsson, Jonas; Olofsson, Sigvard; Bally, Marta; Rydell, Gustaf E

2014-05-01

Studies have suggested that the glycosphingolipid globoside (Gb4Cer) is a receptor for human parvovirus B19. Virus-like particles bind to Gb4Cer on thin-layer chromatograms, but a direct interaction between the virus and lipid membrane-associated Gb4Cer has been debated. Here, we characterized the binding of parvovirus B19 VP1/VP2 virus-like particles to glycosphingolipids (i) on thin-layer chromatograms (TLCs) and (ii) incorporated into supported lipid bilayers (SLBs) acting as cell-membrane mimics. The binding specificities of parvovirus B19 determined in the two systems were in good agreement; the VLP recognized both Gb4Cer and the Forssman glycosphingolipid on TLCs and in SLBs compatible with the role of Gb4Cer as a receptor for this virus. Copyright © 2014 Elsevier Inc. All rights reserved.

Nontrivial behavior of water in the vicinity and inside lipid bilayers as probed by molecular dynamics simulations.

PubMed

Krylov, Nikolay A; Pentkovsky, Vladimir M; Efremov, Roman G

2013-10-22

The atomic-scale diffusion of water in the presence of several lipid bilayers mimicking biomembranes is characterized via unconstrained molecular dynamics (MD) simulations. Although the overall water dynamics corresponds well to literature data, namely, the efficient braking near polar head groups of lipids, a number of interesting and biologically relevant details observed in this work have not been sufficiently discussed so far; for instance, the fact that waters "sense" the membrane unexpectedly early, before water density begins to decrease. In this "transitional zone" the velocity distributions of water and their H-bonding patterns deviate from those in the bulk solution. The boundaries of this zone are well preserved even despite the local (<1 nm size) perturbation of the lipid bilayer, thus indicating a decoupling of the surface and bulk dynamics of water. This is in excellent agreement with recent experimental data. Near the membrane surface, water movement becomes anisotropic, that is, solvent molecules preferentially move outward the bilayer. Deep in the membrane interior, the velocities can even exceed those in the bulk solvent and undergo large-scale fluctuations. The analysis of MD trajectories of individual waters in the middle part of the acyl chain region of lipids reveals a number of interesting rare phenomena, such as the fast (ca. 50 ps) breakthrough across the membrane or long-time (up to 750 ps) "roaming" between lipid leaflets. The analysis of these events was accomplished to delineate the mechanisms of spontaneous water permeation inside the hydrophobic membrane core. It was shown that such nontrivial dynamics of water in an "alien" environment is driven by the dynamic heterogeneities of the local bilayer structure and the formation of transient atomic-scale "defects" in it. The picture observed in lipid bilayers is drastically different from that in a primitive membrane mimic, a hydrated cyclohexane slab. The possible biological impact of such phenomena in equilibrated lipid bilayers is discussed.

Spatial orientation and electric-field-driven transport of hypericin inside of bilayer lipid membranes.

PubMed

Strejčková, Alena; Staničová, Jana; Jancura, Daniel; Miškovský, Pavol; Bánó, Gregor

2013-02-07

Fluorescence experiments were carried out to investigate the interaction of hypericin (Hyp), a natural hydrophobic photosensitizer, with artificial bilayer lipid membranes. The spatial orientation of Hyp monomers incorporated in diphytanoyl phosphatidylcholine (DPhPC) membranes was determined by measuring the dependence of the Hyp fluorescence intensity on the angle of incidence of p- and s-polarized excitation laser beams. Inside of the membrane, Hyp monomers are preferentially located in the layers near the membrane/water interface and are oriented with the S(1) ← S(0) transition dipole moments perpendicular to the membrane surface. Transport of Hyp anions between the two opposite sides of the lipid bilayer was induced by applying rectangular electric field pulses to the membrane. The characteristic time for Hyp transport through the membrane center was evaluated by the analysis of the Hyp fluorescence signal during the voltage pulses. In the zero-voltage limit, the transport time approached 70 ms and gradually decreased with higher voltage applied to the membrane. In addition, our measurements indicated an apparent pK(a) constant of 8 for Hyp deprotonation in the membrane.

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane

PubMed Central

Cathcart, Kelly; Patel, Amit; Dies, Hannah; Rheinstädter, Maikel C.; Fradin, Cécile

2015-01-01

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol’s condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content. PMID:26529029

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane.

PubMed

Cathcart, Kelly; Patel, Amit; Dies, Hannah; Rheinstädter, Maikel C; Fradin, Cécile

2015-10-30

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol's condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content.

Antimicrobial peptides at work: interaction of myxinidin and its mutant WMR with lipid bilayers mimicking the P. aeruginosa and E. coli membranes

NASA Astrophysics Data System (ADS)

Lombardi, Lucia; Stellato, Marco Ignazio; Oliva, Rosario; Falanga, Annarita; Galdiero, Massimiliano; Petraccone, Luigi; D'Errico, Geradino; de Santis, Augusta; Galdiero, Stefania; Del Vecchio, Pompea

2017-03-01

Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armstrong, Clare L; Barrett, M; Heiss, Arno

Inelastic neutron scattering was used to study the effect of 5 and 40 mol% cholesterol on the lateral nanoscale dynamics of phospholipid membranes. By measuring the excitation spectrum at several lateral q || values (up to q || = 3 1), complete dispersion curves were determined of gel, fluid and liquid-ordered phase bilayers. The inclusion of cholesterol had a distinct effect on the collective dynamics of the bilayer s hydrocarbon chains; specifically, we observed a pronounced stiffening of the membranes on the nanometer length scale in both gel and fluid bilayers, even though they were experiencing a higher degree ofmore » molecular disorder. Also, for the first time we determined the nanoscale dynamics in the high-cholesterol liquid-ordered phase of bilayers containing cholesterol. Namely, this phase appears to be softer than fluid bilayers, but better ordered than bilayers in the gel phase.« less

Salt-induced effects on natural and inverse DPPC lipid membranes: Molecular dynamics simulation.

PubMed

Rezaei Sani, Seyed Mojtaba; Akhavan, Mojdeh; Jalili, Seifollah

2018-08-01

Molecular dynamics (MD) simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer and its neutral inverse-phosphocholine equivalent (DPCPe) were performed to find salt-induced effects on their surface structure and the nature of ion-lipid interactions. We found that the area per lipid is not considerably affected by the inversion, but the deuterium order parameter of carbon atoms in the region of carbonyl carbons changes dramatically. MD simulations indicate that Ca 2+ ions can bind to the surface of both DPPC and DPCPe membranes, but K + ions do not bind to them. In the case of Na + , however, the ions can bind to natural lipids but not to the inverse ones. Also, our results demonstrate that the hydration level of CPe bilayers is substantially lower than PC bilayers and the averaged orientation of water dipoles in the region of CPe headgroups is effectively inverted compared to PC lipids. This might be important in the interaction of the bilayer with its biological environment. Furthermore, it was found for the CPe bilayers that the enhanced peaks of the electrostatic potential profiles shift further away from the bilayer center relative to those of PC bilayers. This behavior makes the penetration of cations into the bilayer more difficult and possibly explains the experimentally observed enhanced release rates of anionic compounds in the CPe membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorescent molecular probes based on excited state prototropism in lipid bilayer membrane

NASA Astrophysics Data System (ADS)

Mohapatra, Monalisa; Mishra, Ashok K.

2012-03-01

Excited state prototropism (ESPT) is observed in molecules having one or more ionizable protons, whose proton transfer efficiency is different in ground and excited states. The interaction of various ESPT molecules like naphthols and intramolecular ESPT (ESIPT) molecules like hydroxyflavones etc. with different microheterogeneous media have been studied in detail and excited state prototropism as a probe concept has been gaining ground. The fluorescence of different prototropic forms of such molecules, on partitioning to an organized medium like lipid bilayer membrane, often show sensitive response to the local environment with respect to the local structure, physical properties and dynamics. Our recent work using 1-naphthol as an ESPT fluorescent molecular probe has shown that the incorporation of monomeric bile salt molecules into lipid bilayer membranes composed from dipalmitoylphosphatidylcholine (DPPC, a lung surfactant) and dimyristoylphosphatidylcholine (DMPC), in solid gel and liquid crystalline phases, induce appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (<= 1 mM) concentrations of the bile salts. The incorporation and location of fisetin, an ESIPT molecule having antioxidant properties, in lipid bilayer membrane has been sensitively monitored from its intrinsic fluorescence behaviour.

Control of membrane permeability in air-stable droplet interface bilayers

DOE PAGES

Mruetusatorn, Prachya; Polizos, Georgios; Datskos, Panos G.; ...

2015-03-19

Air-stable droplet interface bilayers (airDIBs) on oil-infused surfaces are versatile model membranes for synthetic biology applications, including biosensing of airborne species. However, air-DIBs are subject to evaporation, which can, over time, destabilize them and reduce their useful lifetime compared to traditional DIBs that are fully submerged in oil. Here, we show that lifetimes of air-DIBs can be extended by as much as an order of magnitude by maintaining them at a temperature just above the dew point. We find that raising the temperature from near the dew point (7 C at 38.5 % relative humidity) to room temperature results inmore » loss of water molecules of hydration from the polar head groups of the lipid bilayer membrane due to evaporation in an irreversible process that increases the overall entropy of the system. This dehydration transition affects primarily the bilayer resistance, by increasing ion permeability through the increasingly disordered polar head group region of the bilayer. Temperature and/or relative humidity are conveniently tunable parameters for controlling the stability and composition of air-DIBs membranes, while still allowing for operation in ambient environments.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rai, Durgesh K.; Sharma, Veerendra K.; Anunciado, Divina

The interaction between lipid bilayers and Amyloid β peptide (Aβ) plays a critical role in proliferation of Alzheimer’s disease (AD). AD is expected to affect one in every 85 humans by 2050, and therefore, deciphering the interplay of Aβ and lipid bilayers at the molecular level is of profound importance. In this work, we applied an array of neutron scattering methods to study the structure and dynamics of Aβ(1–40) interacting 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) bilayers. In the structural investigations of lipid bilayer’s response to Aβ binding, Small Angle Neutron Scattering and Neutron Membrane Diffraction revealed that the Aβ anchors firmly to themore » highly charged DMPG bilayers in the interfacial region between water and hydrocarbon chain, and it doesn’t penetrate deeply into the bilayer. This association mode is substantiated by the dynamics studies with high resolution Quasi-Elastic Neutron Scattering experiments, showing that the addition of Aβ mainly affects the slower lateral motion of lipid molecules, especially in the fluid phase, but not the faster internal motion. The results revealed that Aβ associates with the highly charged membrane in surface with limited impact on the structure, but the altered membrane dynamics could have more influence on other membrane processes.« less

Lipid Raft-Mediated Membrane Tethering and Delivery of Hydrophobic Cargos from Liquid Crystal-Based Nanocarriers.

PubMed

Nag, Okhil K; Naciri, Jawad; Oh, Eunkeu; Spillmann, Christopher M; Delehanty, James B

2016-04-20

A main goal of bionanotechnology and nanoparticle (NP)-mediated drug delivery (NMDD) continues to be the development of novel biomaterials that can controllably modulate the activity of the NP-associated therapeutic cargo. One of the desired subcellular locations for targeted delivery in NMDD is the plasma membrane. However, the controlled delivery of hydrophobic cargos to the membrane bilayer poses significant challenges including cargo precipitation and lack of specificity. Here, we employ a liquid crystal NP (LCNP)-based delivery system for the controlled partitioning of a model dye cargo from within the NP core into the plasma membrane bilayer. During synthesis of the NPs, the water-insoluble model dye cargo, 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), was efficiently incorporated into the hydrophobic LCNP core as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) facilitated the localization of the dye-loaded NPs to lipid raft microdomains in the plasma membrane in HEK 293T/17 cell. Analysis of DiO cellular internalization kinetics revealed that when delivered as a LCNP-PEG-Chol NP, the half-life of DiO membrane residence time (30 min) was twice that of free DiO (DiO(free)) (15 min) delivered from bulk solution. Time-resolved laser scanning confocal microscopy was employed to visualize the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer as confirmed by Förster resonance energy transfer (FRET) imaging. Finally, the delivery of DiO as a LCNP-PEG-Chol complex resulted in the attenuation of its cytotoxicity; the NP form of DiO exhibited ∼30-40% less toxicity compared to DiO(free). Our data demonstrate the utility of the LCNP platform as an efficient vehicle for the combined membrane-targeted delivery and physicochemical modulation of molecular cargos using lipid raft-mediated tethering.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

NASA Astrophysics Data System (ADS)

Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten

2016-09-01

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Preparation of pH-sensitive anionic liposomes designed for drug delivery system (DDS) application.

PubMed

Aoki, Asami; Akaboshi, Hikaru; Ogura, Taku; Aikawa, Tatsuo; Kondo, Takeshi; Tobori, Norio; Yuasa, Makoto

2015-01-01

We prepared pH-sensitive anionic liposomes composed solely of anionic bilayer membrane components that were designed to promote efficient release of entrapped agents in response to acidic pH. The pH-sensitive anionic liposomes showed high dispersion stability at neutral pH, but the fluidity of the bilayer membrane was enhanced in an acidic environment. These liposomes were rather simple and were composed of dimyristoylphosphatidylcholine (DMPC), an anionic bilayer membrane component, and polyoxyethylene sorbitan monostearate (Tween 80). In particular, the present pH-sensitive anionic liposomes showed higher temporal stability than those of conventional DMPC/DPPC liposomes. We found that pHsensitive properties strongly depended on the molecular structure, pKa value, and amount of an incorporated anionic bilayer membrane component, such as sodium oleate (SO), dimyristoylphosphatidylserine (DMPS), or sodium β-sitosterol sulfate (SS). These results provide an opportunity to manipulate liposomal stability in a pH-dependent manner, which could lead to the formulation of a high performance drug delivery system (DDS).

The inner side of T cell lipid rafts.

PubMed

Gri, Giorgia; Molon, Barbara; Manes, Santos; Pozzan, Tullio; Viola, Antonella

2004-07-15

A key question in understanding the functional role of lipid rafts is whether lipid microdomains at the plasma membrane outer leaflet are coupled to lipid microdomains at the inner leaflet. By using a cyan-fluorescent protein (CFP) targeted to inner plasma membrane rafts of Jurkat T cells, we found that raft domains at the outer and inner leaflets are physically coupled and that this coupling requires cholesterol. Interestingly, TCR/CD3 cross-linking induces co-capping of the raft bilayer independently of cholesterol or signaling events, indicating that cholesterol-extracting drugs are unable to destroy TCR-lipid rafts interaction.

Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

NASA Astrophysics Data System (ADS)

Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

2014-07-01

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

Characteristics of TiO2/ZnO bilayer film towards pH sensitivity prepared by different spin coating deposition process

NASA Astrophysics Data System (ADS)

Rahman, Rohanieza Abdul; Zulkefle, Muhammad Al Hadi; Abdullah, Wan Fazlida Hanim; Rusop, M.; Herman, Sukreen Hana

2016-07-01

In this study, titanium dioxide (TiO2) and zinc oxide (ZnO) bilayer film for pH sensing application will be presented. TiO2/ZnO bilayer film with different speed of spin-coating process was deposited on Indium Tin Oxide (ITO), prepared by sol-gel method. This fabricated bilayer film was used as sensing membrane for Extended Gate Field-Effect Transistor (EGFET) for pH sensing application. Experimental results indicated that the sensor is able to detect the sensitivity towards pH buffer solution. In order to obtained the result, sensitivity measurement was done by using the EGFET setup equipment with constant-current (100 µA) and constant-voltage (0.3 V) biasing interfacing circuit. TiO2/ZnO bilayer film which the working electrode, act as the pH-sensitive membrane was connected to a commercial metal-oxide semiconductor FET (MOSFET). This MOSFET then was connected to the interfacing circuit. The sensitivity of the TiO2 thin film towards pH buffer solution was measured by dipping the sensing membrane in pH4, pH7 and pH10 buffer solution. These thin films were characterized by using Field Emission Scanning Electron Microscope (FESEM) to obtain the surface morphology of the composite bilayer films. In addition, I-V measurement was done in order to determine the electrical properties of the bilayer films. According to the result obtained in this experiment, bilayer film that spin at 4000 rpm, gave highest sensitivity which is 52.1 mV/pH. Relating the I-V characteristic of the thin films and sensitivity, the sensing membrane with higher conductivity gave better sensitivity.

Characteristics of TiO{sub 2}/ZnO bilayer film towards pH sensitivity prepared by different spin coating deposition process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahman, Rohanieza Abdul, E-mail: rohanieza.abdrahman@gmail.com; Zulkefle, Muhammad Al Hadi, E-mail: alhadizulkefle@gmail.com; Abdullah, Wan Fazlida Hanim, E-mail: wanfaz@salam.uitm.edu.my

In this study, titanium dioxide (TiO{sub 2}) and zinc oxide (ZnO) bilayer film for pH sensing application will be presented. TiO{sub 2}/ZnO bilayer film with different speed of spin-coating process was deposited on Indium Tin Oxide (ITO), prepared by sol-gel method. This fabricated bilayer film was used as sensing membrane for Extended Gate Field-Effect Transistor (EGFET) for pH sensing application. Experimental results indicated that the sensor is able to detect the sensitivity towards pH buffer solution. In order to obtained the result, sensitivity measurement was done by using the EGFET setup equipment with constant-current (100 µA) and constant-voltage (0.3 V)more » biasing interfacing circuit. TiO{sub 2}/ZnO bilayer film which the working electrode, act as the pH-sensitive membrane was connected to a commercial metal-oxide semiconductor FET (MOSFET). This MOSFET then was connected to the interfacing circuit. The sensitivity of the TiO2 thin film towards pH buffer solution was measured by dipping the sensing membrane in pH4, pH7 and pH10 buffer solution. These thin films were characterized by using Field Emission Scanning Electron Microscope (FESEM) to obtain the surface morphology of the composite bilayer films. In addition, I-V measurement was done in order to determine the electrical properties of the bilayer films. According to the result obtained in this experiment, bilayer film that spin at 4000 rpm, gave highest sensitivity which is 52.1 mV/pH. Relating the I-V characteristic of the thin films and sensitivity, the sensing membrane with higher conductivity gave better sensitivity.« less

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

PubMed Central

Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. PMID:25106608

Insertion and self-diffusion of a monotopic protein, the Aquifex aeolicus sulfide quinone reductase, in supported lipid bilayers.

PubMed

Harb, Frédéric; Prunetti, Laurence; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne; Tinland, Bernard

2015-10-01

Monotopic proteins constitute a class of membrane proteins that bind tightly to cell membranes, but do not span them. We present a FRAPP (Fluorescence Recovery After Patterned Photobleaching) study of the dynamics of a bacterial monotopic protein, SQR (sulfide quinone oxidoreductase) from the thermophilic bacteria Aquifex aeolicus, inserted into two different types of lipid bilayers (EggPC: L-α-phosphatidylcholine (Egg, Chicken) and DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine) supported on two different types of support (mica or glass). It sheds light on the behavior of a monotopic protein inside the bilayer. The insertion of SQR is more efficient when the bilayer is in the fluid phase than in the gel phase. We observed diffusion of the protein, with no immobile fraction, and deduced from the diffusion coefficient measurements that the resulting inserted object is the same whatever the incubation conditions, i.e. homogeneous in terms of oligomerization state. As expected, the diffusion coefficient of the SQR is smaller in the gel phase than in the fluid phase. In the supported lipid bilayer, the diffusion coefficient of the SQR is smaller than the diffusion coefficient of phospholipids in both gel and fluid phase. SQR shows a diffusion behavior different from the transmembrane protein α-hemolysin, and consistent with its monotopic character. Preliminary experiments in the presence of the substrate of SQR, DecylUbiquinone, an analogue of quinone, component of transmembrane electrons transport systems of eukaryotic and prokaryotic organisms, have been carried out. Finally, we studied the behavior of SQR, in terms of insertion and diffusion, in bilayers formed with lipids from Aquifex aeolicus. All the conclusions that we have found in the biomimetic systems applied to the biological system.

Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

PubMed Central

2007-01-01

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 μm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane. PMID:17065557

Liver mitochondrial membrane fluidity at early development of diabetes and its correlation with the respiration.

PubMed

Pérez-Hernández, Ismael H; Domínguez-Fuentes, Josué Misael; Palomar-Morales, Martín; Zazueta-Mendizabal, Ana Cecilia; Baiza-Gutman, Arturo; Mejía-Zepeda, Ricardo

2017-06-01

The biological membranes are important in cell function but, during development of diseases such as diabetes, they are impaired. Consequently, membrane-associated biological processes are impaired as well. The mitochondria are important organelles where oxidative phosphorylation takes place, a process closely related with the membranes. In general, it is accepted that the development process of diabetes decreases membrane fluidity. However, in some cases, it has been found to increase membrane fluidity of mitochondria but to decrease the Respiratory Control (RC) index. In this study we found an increase of membrane fluidity and an increase of the RC at an early phase of the development of a type 2 diabetes model. We measured the lipoperoxidation, analyzed the fatty acids composition by gas chromatography, and assessed membrane fluidity using three fluorescent monitors located at different depths inside the bilayer, dipyrenilpropane (DPyP), diphenylhexatriene (DPH), and trimethylammonium diphenylhexatriene (TMA-DPH). Our findings indicate that in the initial stage of diabetes development, when lipoperoxidation still is not significant, the membrane fluidity of liver mitochondria increases because of the increment in the unsaturated to saturated fatty acids ratio (U/S), thus producing an increase of the RC. The membrane fluidity is not the same at all depths in the bilayer. Contrary to the results obtained in mitochondria, the diabetes induced a decrease in the U/S fatty acids ratio of liver total lipids, indicating that the mitochondria might have an independent mechanism for regulating its fatty acids composition.

HIV and Drug Resistance: Hitting a Moving Target | Center for Cancer Research

Cancer.gov

Prior research revealed how HIV-1 makes its destructive entry into the target cell by fusing together the cholesterol-rich lipid bilayer of the viral envelope—made with key glycoproteins gp120 and gp41—and the host cell’s plasma membrane. Cell-viral interactions begin with the binding of gp120 to the CD4 receptor molecule on the target cell, followed by gp120 binding to

Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

PubMed

Ghang, Yoo-Jin; Perez, Lizeth; Morgan, Melissa A; Si, Fang; Hamdy, Omar M; Beecher, Consuelo N; Larive, Cynthia K; Julian, Ryan R; Zhong, Wenwan; Cheng, Quan; Hooley, Richard J

2014-12-28

An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface.

Mn2+ exerts stronger structural effects than the Mn-citrate complex on the human erythrocyte membrane and molecular models.

PubMed

Suwalsky, M; Villena, F; Sotomayor, C P

2010-01-01

While traces of manganese (Mn) take part in important and essential functions in biology, elevated exposures have been shown to cause significant toxicity. Chronic exposure to the metal leads to manganese neurotoxicity (or manganism), a brain disorder that resembles Parkinsonism. Toxic effect mechanisms of Mn is not understood, toxic concentrations of manganese are not well defined and blood manganese concentration at which neurotoxicity occurs has not been identified. There are reports indicating that the most abundant Mn-species in Mn carriers within blood is the Mn-citrate complex. Despite the well-documented information about the toxic effects of Mn, there are scarce reports concerning the effects of manganese compounds on both structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of Mn with cell membranes, MnCl(2), and the Mn-citrate complex were incubated with intact erythrocytes, isolated unsealead human erythrocyte membranes (IUM), and molecular models of the erythrocyte membrane. These consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of the erythrocyte membrane, respectively. The capacity of the Mn compounds to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). In all these systems it was found that Mn(2+) exerted considerable higher structural perturbations than the Mn-citrate complex.

Structure refinement of membrane proteins via molecular dynamics simulations.

PubMed

Dutagaci, Bercem; Heo, Lim; Feig, Michael

2018-07-01

A refinement protocol based on physics-based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge-based or implicit membrane-based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid-facing residues. Scoring with knowledge-based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane-based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models. © 2018 Wiley Periodicals, Inc.

Structure and hydration of membranes embedded with voltage-sensing domains.

PubMed

Krepkiy, Dmitriy; Mihailescu, Mihaela; Freites, J Alfredo; Schow, Eric V; Worcester, David L; Gawrisch, Klaus; Tobias, Douglas J; White, Stephen H; Swartz, Kenton J

2009-11-26

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.

Structure and hydration of membranes embedded with voltage-sensing domains

PubMed Central

Krepkiy, Dmitriy; Mihailescu, Mihaela; Freites, J. Alfredo; Schow, Eric V.; Worcester, David L.; Gawrisch, Klaus; Tobias, Douglas; White, Stephen H.; Swartz, Kenton J.

2009-01-01

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly-charged S1–S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated potassium channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1–S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field. PMID:19940918

Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu

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 (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RCmore » within the tethered 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.« less

Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum.

PubMed

Takahashi, Kei; Toyota, Taro

2017-03-07

The transformation of the supported lipid bilayer (SLB) membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB) membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum , is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level), contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane.

Functional membranes. Present and future

NASA Technical Reports Server (NTRS)

Kunitake, T.

1982-01-01

The present situation and the future development of the functional membrane are discussed. It is expected that functional membranes will play increasingly greater roles in the chemical industry of the coming decade. These membranes are formed from polymer films, liquid membranes or bilayer membranes. The two most important technologies based on the polymeric membrane are reverse osmosis and ion exchange. The liquid membrane is used for separation of ionic species; an extension of the solvent extraction process. By using appropriate ligands and ionophores, highly selective separations are realized. The active transport is made possible if the physical and chemical potentials are applied to the transport process. More advanced functional membranes may be designed on the basis of the synthetic bilayer membrane.

Planar patch clamp: advances in electrophysiology.

PubMed

Brüggemann, Andrea; Farre, Cecilia; Haarmann, Claudia; Haythornthwaite, Ali; Kreir, Mohamed; Stoelzle, Sonja; George, Michael; Fertig, Niels

2008-01-01

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes*

PubMed Central

Zhou, Yong; Plowman, Sarah J.; Lichtenberger, Lenard M.; Hancock, John F.

2010-01-01

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase seperation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane. PMID:20826816

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

PubMed Central

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.

2013-01-01

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. PMID:23947891

Applications of solid-state NMR to membrane proteins.

PubMed

Ladizhansky, Vladimir

2017-11-01

Membrane proteins mediate flow of molecules, signals, and energy between cells and intracellular compartments. Understanding membrane protein function requires a detailed understanding of the structural and dynamic properties involved. Lipid bilayers provide a native-like environment for structure-function investigations of membrane proteins. In this review we give a general discourse on the recent progress in the field of solid-state NMR of membrane proteins. Solid-state NMR is a variation of NMR spectroscopy that is applicable to molecular systems with restricted mobility, such as high molecular weight proteins and protein complexes, supramolecular assemblies, or membrane proteins in a phospholipid environment. We highlight recent advances in applications of solid-state NMR to membrane proteins, specifically focusing on the recent developments in the field of Dynamic Nuclear Polarization, proton detection, and solid-state NMR applications in situ (in cell membranes). This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding.

PubMed

Kleinschmidt, J H; Tamm, L K

1999-04-20

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.

Delivery of nitric oxide to the interior of mammalian cell by carbon nanotube: MD simulation.

PubMed

Raczyński, Przemysław; Górny, Krzysztof; Dawid, Aleksander; Gburski, Zygmunt

2014-07-15

Computer simulations have been performed to study the nanoindentation of phospholipid bilayer by the single-walled armchair carbon nanotube, filled with the nitric oxide molecules. The process has been simulated by means of molecular dynamics (MD) technique at physiological temperature T = 310 K with a constant pulling velocity of the nanotube. The force acting on the nanotube during membrane penetration has been calculated. We show that the indentation by carbon nanotube does not permanently destroy the membrane structure (self-sealing of the membrane occurs). The mobility of nitric oxide molecules during the membrane nanoindentation is discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

PubMed

Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

2014-03-01

Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

PubMed Central

Verstraeten, Sandra V; Zago, M Paola; MacKenzie, Gerardo G; Keen, Carl L; Oteiza, Patricia I

2004-01-01

We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution. PMID:14629198