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Sample records for lipid membranes determined

  1. Determining the pivotal plane of fluid lipid membranes in simulations

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Deserno, Markus

    2015-10-01

    Each leaflet of a curved lipid membrane contains a surface at which the area strain vanishes, the so-called pivotal plane. Its distance z0 from the bilayer's midplane arises in numerous contexts, for instance the connection between monolayer and bilayer moduli, stress-profile moments, or area-difference elasticity theories. Here, we propose two precise methods for determining the location of the pivotal plane in computer simulations, both of which rely on monitoring the lipid imbalance across a curved bilayer. The first method considers the ratio of lipid number between the two leaflets of cylindrical or spherical vesicles; it hence requires lipid flip-flop for equilibration. The second method looks at the leaflet difference across local sections cut out from a buckled membrane; this observable equilibrates even in the absence of flip-flop. We apply our methods to two different coarse-grained lipid models, the generic three-bead solvent-free Cooke model and a ten-bead representation of dimyristoylphosphocholine with the explicit solvent MARTINI model. The Cooke model is amenable to both methods and gives results that agree at the percent level. Using it, we also show that the pivotal plane moves outward as lipid curvature becomes more positive. The MARTINI model can only be analyzed with the buckling method; the obtained value z0 = 0.850(11) nm lies about 0.4 nm inwards of the glycerol backbone and is hence unexpectedly small. We attribute this to limitations of the coarse-grained description, suggesting that the location of the pivotal plane might be a good indicator for how well lipid models capture the microscopic origins of curvature elasticity. Finally, we also show that the pivotal plane position itself moves as the membrane is bent. The leading correction is linear in curvature, dependent on the Poisson ratio, and can matter when analyzing experimental results obtained from highly curved inverse hexagonal phases.

  2. Lipid bilayer thickness determines cholesterol's location in model membranes

    SciTech Connect

    Marquardt, Drew; Heberle, Frederick A.; Greathouse, Denise V.; Koeppe, II, Roger E.; Standaert, Robert F.; Van Oosten, Brad J.; Harroun, Thad A.; Kinnun, Jacob J.; Williams, Justin A.; Wassall, Stephen R.; Katsaras, John

    2016-10-11

    Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of different lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. Finally, these results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.

  3. Lipid bilayer thickness determines cholesterol's location in model membranes

    DOE PAGES

    Marquardt, Drew; Heberle, Frederick A.; Greathouse, Denise V.; ...

    2016-10-11

    Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of differentmore » lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. Finally, these results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.« less

  4. Introduction to membrane lipids.

    PubMed

    Epand, Richard M

    2015-01-01

    Biological membranes are composed largely of lipids and proteins. The most common arrangement of lipids in biological membranes is as a bilayer. This arrangement spontaneously forms a barrier for the passage of polar materials. The bilayer is thin but can have a large area in the dimension perpendicular to its thickness. The physical nature of the bilayer membrane will vary according to the conditions of the environment as well as the chemical structure of the lipid constituents of the bilayer. These physical properties determine the function of the membrane together with specific structural features of the lipids that allow them to have signaling properties. The lipids of the membrane are not uniformly distributed. There is an intrinsic asymmetry between the two monolayers that constitute the bilayer. In addition, some lipids tend to be enriched in particular regions of the membrane, termed domains. There is evidence that certain domains recruit specific proteins into that domain. This has been suggested to be important for allowing interaction among different proteins involved in certain signal transduction pathways. Membrane lipids have important roles in determining the physical properties of the membrane, in modulating the activity of membrane-bound proteins and in certain cases being specific secondary messengers that can interact with specific proteins. A large variety of lipids present in biological membranes result in them possessing many functions.

  5. Lipids that determine detergent resistance of MDCK cell membrane fractions.

    PubMed

    Manni, Marco M; Cano, Ainara; Alonso, Cristina; Goñi, Félix M

    2015-10-01

    A comparative lipidomic study has been performed of whole Madin-Darby canine kidney epithelial cells and of the detergent-resistant membrane fraction (DRM) obtained after treating the cells with the non-ionic detergent Triton X-100. The DRM were isolated following a standard procedure that is extensively used in cell biology studies. Significant differences were found in the lipid composition of the whole cells and of DRM. The latter were enriched in all the analyzed sphingolipid classes: sphingomyelins, ceramides and hexosylceramides. Diacylglycerols were also preferentially found in DRM. The detergent-resistant fraction was also enriched in saturated over unsaturated fatty acyl chains, and in sn-1 acyl chains containing 16 carbon atoms, over the longer and shorter ones. The glycerophospholipid species phosphatidylethanolamines and phosphatidylinositols, that were mainly unsaturated, did not show a preference for DRM. Phosphatidylcholines were an intermediate case: the saturated, but not the unsaturated species were found preferentially in DRM. The question remains on whether these DRM, recovered from detergent-membrane mixtures by floatation over a sucrose gradient, really correspond to membrane domains existing in the cell membrane prior to detergent treatment.

  6. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    PubMed

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  7. Structural determinants of protein partitioning into ordered membrane domains and lipid rafts.

    PubMed

    Lorent, Joseph Helmuth; Levental, Ilya

    2015-11-01

    Increasing evidence supports the existence of lateral nanoscopic lipid domains in plasma membranes, known as lipid rafts. These domains preferentially recruit membrane proteins and lipids to facilitate their interactions and thereby regulate transmembrane signaling and cellular homeostasis. The functionality of raft domains is intrinsically dependent on their selectivity for specific membrane components; however, while the physicochemical determinants of raft association for lipids are known, very few systematic studies have focused on the structural aspects that guide raft partitioning of proteins. In this review, we describe biophysical and thermodynamic aspects of raft-mimetic liquid ordered phases, focusing on those most relevant for protein partitioning. Further, we detail the variety of experimental models used to study protein-raft interactions. Finally, we review the existing literature on mechanisms for raft targeting, including lipid post-translational modifications, lipid binding, and transmembrane domain features. We conclude that while protein palmitoylation is a clear raft-targeting signal, few other general structural determinants for raft partitioning have been revealed, suggesting that many discoveries lie ahead in this burgeoning field.

  8. Lipids and Membrane Lateral Organization

    PubMed Central

    Sonnino, Sandro; Prinetti, Alessandro

    2010-01-01

    Shortly after the elucidation of the very basic structure and properties of cellular membranes, it became evident that cellular membranes are highly organized structures with multiple and multi-dimensional levels of order. Very early observations suggested that the lipid components of biological membranes might be active players in the creation of these levels of order. In the late 1980s, several different and diverse experimental pieces of evidence coalesced together giving rise to the lipid raft hypothesis. Lipid rafts became enormously (and, in the opinion of these authors, sometimes acritically) popular, surprisingly not just within the lipidologist community (who is supposed to be naturally sensitive to the fascination of lipid rafts). Today, a PubMed search using the key word “lipid rafts” returned a list of 3767 papers, including 690 reviews (as a term of comparison, searching over the same time span for a very hot lipid-related key word, “ceramide” returned 6187 hits with 799 reviews), and a tremendous number of different cellular functions have been described as “lipid raft-dependent.” However, a clear consensus definition of lipid raft has been proposed only in recent times, and the basic properties, the ruling forces, and even the existence of lipid rafts in living cells has been recently matter of intense debate. The scenario that is gradually emerging from the controversies elicited by the lipid raft hypothesis emphasizes multiple roles for membrane lipids in determining membrane order, that encompass their tendency to phase separation but are clearly not limited to this. In this review, we would like to re-focus the attention of the readers on the importance of lipids in organizing the fine structure of cellular membranes. PMID:21423393

  9. Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence-activated flow cytometry analyses were developed for determination of reactive oxygen species (ROS) formation and membrane lipid peroxidation in live spermatozoa loaded with, respectively, hydroethidine (HE) or the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-d...

  10. Anionic Lipids: Determinants of Binding Cytotoxins from Snake Venom on the Surface of Cell Membranes

    PubMed Central

    Boldyrev, I.A.; Omelkov, A.V.; Utkin, Yu.N.; Efremov, R.G.

    2010-01-01

    The cytotoxic properties of cytotoxins (CTs) from snake venom are mediated by their interaction with the cell membrane. The hydrophobic pattern containing the tips of loops I–III and flanked by polar residues is known to be a membrane–binding motif of CTs. However, this is not enough to explain the difference in activity among various CTs which are similar in sequence and in 3D structure. The mechanism of further CT–membrane interaction leading to pore formation and cell death still remains unknown. Published experimental data on the specific interaction between CT and low molecular weight anionic components (sulphatide) of the bilayer point to the existence of corresponding ligand binding sites on the surface of toxin molecules. In this work we study the membrane–lytic properties of CT I, CT II (Naja oxiana), and Ct 4 (Naja kaouthia), which belong to different structural and functional types (P– and S–type) of CTs, by measuring the intensity of a fluorescent dye, calcein released from liposomes containing a phosphatidylserine (PS) lipid as an anionic component. Using molecular docking simulations, we find and characterize three sites in CT molecules that can potentially bind the PS polar head. Based on the data obtained, we suggest a hypothesis that CTs can specifically interact with one or more of the anionic lipids (in particular, with PS) contained in the membrane, thus facilitating the interaction between CTs and the lipid bilayer of a cell membrane. PMID:22649646

  11. Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method

    SciTech Connect

    Li, Dianfan; Pye, Valerie E.; Caffrey, Martin

    2015-01-01

    Very little information is available in the literature concerning the experimental heavy-atom phasing of membrane-protein structures where the crystals have been grown using the lipid cubic phase (in meso) method. In this paper, pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine labelling as applied to an integral membrane kinase crystallized in meso are described. An assay to assess cysteine accessibility for mercury labelling of membrane proteins is introduced. Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.

  12. Milk fat content and DGAT1 genotype determine lipid composition of the milk fat globule membrane.

    PubMed

    Argov-Argaman, Nurit; Mida, Kfir; Cohen, Bat-Chen; Visker, Marleen; Hettinga, Kasper

    2013-01-01

    During secretion of milk fat globules, triacylglycerol (TAG) droplets are enveloped by a phospholipid (PL) trilayer. Globule size has been found to be related to polar lipid composition and fat content, and milk fat content and fatty acid composition have been associated with the diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism; however, the association between the DGAT1 polymorphism and fat globule size and polar lipid composition has not been studied. The ratio between polar and neutral lipids as well as the composition of the polar lipids in milk has industrial as well as nutritional and health implications. Understanding phenotypic and genotypic factors influencing these parameters could contribute to improving milk lipid composition for dairy products. The focus of the present study was to determine the effect of both fat content and DGAT1 polymorphism on PL/TAG ratio, as a marker for milk fat globule size, and detailed PL composition. Milk samples were selected from 200 cows such that there were equal numbers of samples for the different fat contents as well as per DGAT1 genotype. Samples were analyzed for neutral and polar lipid concentration and composition. PL/TAG ratio was significantly associated with both fat content and DGAT1 genotype. Phosphatidylinositol and phosphatidylserine concentrations were associated with fat content*DGAT1 genotype with a stronger association for the AA than the KK genotype. Sphingomyelin concentration tended to interact with fat content*DGAT1 genotype. Phosphatidylethanolamine (PE) concentration showed a biphasic response to fat content, suggesting that multiple biological processes influence its concentration. These results provide a new direction for controlling polar lipid concentration and composition in milk through selective breeding of cows.

  13. Characterization of lipid domains in erythrocyte membranes.

    PubMed

    Rodgers, W; Glaser, M

    1991-02-15

    Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.

  14. Dynamics of multicomponent lipid membranes

    NASA Astrophysics Data System (ADS)

    Camley, Brian Andrew

    We present theoretical and computational descriptions of the dynamics of multicomponent lipid bilayer membranes. These systems are both model systems for "lipid rafts" in cell membranes and interesting physical examples of quasi-two-dimensional fluids. Our chief tool is a continuum simulation that uses a phase field to track the composition of the membrane, and solves the hydrodynamic equations exactly using the appropriate Green's function (Oseen tensor) for the membrane. We apply this simulation to describe the diffusion of domains in phase-separated membranes, the dynamics of domain flickering, and the process of phase separation in lipid membranes. We then derive an analytical theory to describe domain flickering that is consistent with our simulation results, and use this to analyze experimental measurements of membrane domains. Through this method, we measure the membrane viscosity solely from fluorescence microscopy measurements. We study phase separation in quasi-two-dimensional membranes in depth with both simulations and scaling theory, and classify the different scaling regimes and morphologies, which differ from pure two-dimensional fluids. Our results may explain previous inconsistent measurements of the dynamical scaling exponent for phase separation in membranes. We also extend our theory beyond the simplest model, including the possibility that the membrane will be viscoelastic, as well as considering the inertia of the membrane and the fluid surrounding the membrane.

  15. Electronic polymers in lipid membranes.

    PubMed

    Johansson, Patrik K; Jullesson, David; Elfwing, Anders; Liin, Sara I; Musumeci, Chiara; Zeglio, Erica; Elinder, Fredrik; Solin, Niclas; Inganäs, Olle

    2015-06-10

    Electrical interfaces between biological cells and man-made electrical devices exist in many forms, but it remains a challenge to bridge the different mechanical and chemical environments of electronic conductors (metals, semiconductors) and biosystems. Here we demonstrate soft electrical interfaces, by integrating the metallic polymer PEDOT-S into lipid membranes. By preparing complexes between alkyl-ammonium salts and PEDOT-S we were able to integrate PEDOT-S into both liposomes and in lipid bilayers on solid surfaces. This is a step towards efficient electronic conduction within lipid membranes. We also demonstrate that the PEDOT-S@alkyl-ammonium:lipid hybrid structures created in this work affect ion channels in the membrane of Xenopus oocytes, which shows the possibility to access and control cell membrane structures with conductive polyelectrolytes.

  16. Lipid Polymorphisms and Membrane Shape

    PubMed Central

    Frolov, Vadim A.; Shnyrova, Anna V.; Zimmerberg, Joshua

    2011-01-01

    Morphological plasticity of biological membrane is critical for cellular life, as cells need to quickly rearrange their membranes. Yet, these rearrangements are constrained in two ways. First, membrane transformations may not lead to undesirable mixing of, or leakage from, the participating cellular compartments. Second, membrane systems should be metastable at large length scales, ensuring the correct function of the particular organelle and its turnover during cellular division. Lipids, through their ability to exist with many shapes (polymorphism), provide an adequate construction material for cellular membranes. They can self-assemble into shells that are very flexible, albeit hardly stretchable, which allows for their far-reaching morphological and topological behaviors. In this article, we will discuss the importance of lipid polymorphisms in the shaping of membranes and its role in controlling cellular membrane morphology. PMID:21646378

  17. Lipid membranes on nanostructured silicon.

    SciTech Connect

    Slade, Andrea Lynn; Lopez, Gabriel P.; Ista, Linnea K.; O'Brien, Michael J.; Sasaki, Darryl Yoshio; Bisong, Paul; Zeineldin, Reema R.; Last, Julie A.; Brueck, Stephen R. J.

    2004-12-01

    A unique composite nanoscale architecture that combines the self-organization and molecular dynamics of lipid membranes with a corrugated nanotextured silicon wafer was prepared and characterized with fluorescence microscopy and scanning probe microscopy. The goal of this project was to understand how such structures can be assembled for supported membrane research and how the interfacial interactions between the solid substrate and the soft, self-assembled material create unique physical and mechanical behavior through the confinement of phases in the membrane. The nanometer scale structure of the silicon wafer was produced through interference lithography followed by anisotropic wet etching. For the present study, a line pattern with 100 nm line widths, 200 nm depth and a pitch of 360 nm pitch was fabricated. Lipid membranes were successfully adsorbed on the structured silicon surface via membrane fusion techniques. The surface topology of the bilayer-Si structure was imaged using in situ tapping mode atomic force microscopy (AFM). The membrane was observed to drape over the silicon structure producing an undulated topology with amplitude of 40 nm that matched the 360 nm pitch of the silicon structure. Fluorescence recovery after photobleaching (FRAP) experiments found that on the microscale those same structures exhibit anisotropic lipid mobility that was coincident with the silicon substructure. The results showed that while the lipid membrane maintains much of its self-assembled structure in the composite architecture, the silicon substructure indeed influences the dynamics of the molecular motion within the membrane.

  18. 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.

  19. Disposition of ceramide in model lipid membranes determined by neutron diffraction.

    PubMed

    Groen, D; Gooris, G S; Barlow, D J; Lawrence, M J; van Mechelen, J B; Demé, B; Bouwstra, J A

    2011-03-16

    The lipid matrix present in the uppermost layer of the skin, the stratum corneum, plays a crucial role in the skin barrier function. The lipids are organized into two lamellar phases. To gain more insight into the molecular organization of one of these lamellar phases, we performed neutron diffraction studies. In the diffraction pattern, five diffraction orders were observed attributed to a lamellar phase with a repeat distance of 5.4 nm. Using contrast variation, the scattering length density profile could be calculated showing a typical bilayer arrangement. To obtain information on the arrangement of ceramides in the unit cell, a mixture that included a partly deuterated ceramide was also examined. The scattering length density profile of the 5.4-nm phase containing this deuterated ceramide demonstrated a symmetric arrangement of the ceramides with interdigitating acyl chains in the center of the unit cell.

  20. Film Balance Studies of Membrane Lipids and Related Molecules

    ERIC Educational Resources Information Center

    Cadenhead, D. A.

    1972-01-01

    Discusses apparatus, techniques, and measurements used to determine cell membrane composition. The use of a film balance to study monolayer membranes of selected lipids is described and results reported. (TS)

  1. FCS diffusion laws in two-phase lipid membranes: determination of domain mean size by experiments and Monte Carlo simulations.

    PubMed

    Favard, Cyril; Wenger, Jérôme; Lenne, Pierre-François; Rigneault, Hervé

    2011-03-02

    Many efforts have been undertaken over the last few decades to characterize the diffusion process in model and cellular lipid membranes. One of the techniques developed for this purpose, fluorescence correlation spectroscopy (FCS), has proved to be a very efficient approach, especially if the analysis is extended to measurements on different spatial scales (referred to as FCS diffusion laws). In this work, we examine the relevance of FCS diffusion laws for probing the behavior of a pure lipid and a lipid mixture at temperatures below, within and above the phase transitions, both experimentally and numerically. The accuracy of the microscopic description of the lipid mixtures found here extends previous work to a more complex model in which the geometry is unknown and the molecular motion is driven only by the thermodynamic parameters of the system itself. For multilamellar vesicles of both pure lipid and lipid mixtures, the FCS diffusion laws recorded at different temperatures exhibit large deviations from pure Brownian motion and reveal the existence of nanodomains. The variation of the mean size of these domains with temperature is in perfect correlation with the enthalpy fluctuation. This study highlights the advantages of using FCS diffusion laws in complex lipid systems to describe their temporal and spatial structure.

  2. Photoinduced Fusion of Lipid Bilayer Membranes.

    PubMed

    Suzuki, Yui; Nagai, Ken H; Zinchenko, Anatoly; Hamada, Tsutomu

    2017-03-14

    We have developed a novel system for photocontrol of the fusion of lipid vesicles through the use of a photosensitive surfactant containing an azobenzene moiety (AzoTAB). Real-time microscopic observations clarified a change in both the surface area and internal volume of vesicles during fusion. We also determined the optimal cholesterol concentrations and temperature for inducing fusion. The mechanism of fusion can be attributed to a change in membrane tension, which is caused by the solubilization of lipids through the isomerization of AzoTAB. We used a micropipet technique to estimate membrane tension and discuss the mechanism of fusion in terms of membrane elastic energy. The obtained results regarding this novel photoinduced fusion could lead to a better understanding of the mechanism of membrane fusion in living cells and may also see wider applications, such as in drug delivery and biomimetic material design.

  3. MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

    PubMed Central

    Stansfeld, Phillip J.; Goose, Joseph E.; Caffrey, Martin; Carpenter, Elisabeth P.; Parker, Joanne L.; Newstead, Simon; Sansom, Mark S.P.

    2015-01-01

    Summary There has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein. PMID:26073602

  4. Lipid membrane domains in the brain.

    PubMed

    Aureli, Massimo; Grassi, Sara; Prioni, Simona; Sonnino, Sandro; Prinetti, Alessandro

    2015-08-01

    The brain is characterized by the presence of cell types with very different functional specialization, but with the common trait of a very high complexity of structures originated by their plasma membranes. Brain cells bear evident membrane polarization with the creation of different morphological and functional subcompartments, whose formation, stabilization and function require a very high level of lateral order within the membrane. In other words, the membrane specialization of brain cells implies the presence of distinct membrane domains. The brain is the organ with the highest enrichment in lipids like cholesterol, glycosphingolipids, and the most recently discovered brain membrane lipid, phosphatidylglucoside, whose collective behavior strongly favors segregation within the membrane leading to the formation of lipid-driven membrane domains. Lipid-driven membrane domains function as dynamic platforms for signal transduction, protein processing, and membrane turnover. Essential events involved in the development and in the maintenance of the functional integrity of the brain depend on the organization of lipid-driven membrane domains, and alterations in lipid homeostasis, leading to deranged lipid-driven membrane organization, are common in several major brain diseases. In this review, we summarize the forces behind the formation of lipid membrane domains and their biological roles in different brain cells. This article is part of a Special Issue entitled Brain Lipids.

  5. Determination of the dissociation constants for Ca2+ and calmodulin from the plasma membrane Ca2+ pump by a lipid probe that senses membrane domain changes.

    PubMed

    Mangialavori, Irene; Ferreira-Gomes, Mariela; Pignataro, María F; Strehler, Emanuel E; Rossi, Juan Pablo F C

    2010-01-01

    The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

  6. Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.

    PubMed

    Li, Dianfan; Boland, Coilín; Aragao, David; Walsh, Kilian; Caffrey, Martin

    2012-09-02

    An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases(1-5), has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field(6-21) (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting(22,23). Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)(24,25) are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection. The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies(4,26). The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been

  7. Polymer Diffusion in Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Prasad, Ashok

    2005-03-01

    Motivated by experiments on fluorescently labeled DNA molecules on a supported lipid bilayer, we have examined theoretically diffusion of polymers in two dimensions. The key experimental finding we focus on is the scaling of the diffusion constant of the center of mass, D˜1/N. This implies that no effective hydrodynamic coupling exists between the diffusing DNA segments in the membrane. We construct our theoretical model using the phenomenological hydrodynamic model of supported membranes proposed by Evans and Sackmann. Our model is based on the pre-averaged Oseen tensor, and is similar to the model of Komura and Seki, but elaborated and extended to take explicit account of self-avoidance. We find that the 1/N scaling of D can be understood as a consequence of membrane hydrodynamics in the presence of a supporting surface. Further experimental consequences of the model, in particular the diffusion constant for DNA in free standing membranes, will also be discussed. This work was supported by the NSF through grants DMR-9984471 and DMR-0403997. JK is a Cottrell Scholar of Research Corporation.

  8. Atomic force microscopy of model lipid membranes.

    PubMed

    Morandat, Sandrine; Azouzi, Slim; Beauvais, Estelle; Mastouri, Amira; El Kirat, Karim

    2013-02-01

    Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir-Blodgett or Langmuir-Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

  9. Brain membrane lipids in major depression and anxiety disorders.

    PubMed

    Müller, Christian P; Reichel, Martin; Mühle, Christiane; Rhein, Cosima; Gulbins, Erich; Kornhuber, Johannes

    2015-08-01

    Major depression and anxiety disorders have high prevalence rates and are frequently comorbid. The neurobiological bases for these disorders are not fully understood, and available treatments are not always effective. Current models assume that dysfunctions in neuronal proteins and peptide activities are the primary causes of these disorders. Brain lipids determine the localization and function of proteins in the cell membrane and in doing so regulate synaptic throughput in neurons. Lipids may also leave the membrane as transmitters and relay signals from the membrane to intracellular compartments or to other cells. Here we review how membrane lipids, which play roles in the membrane's function as a barrier and a signaling medium for classical transmitter signaling, contribute to depression and anxiety disorders and how this role may provide targets for lipid-based treatment approaches. Preclinical findings have suggested a crucial role for the membrane-forming n-3 polyunsaturated fatty acids, glycerolipids, glycerophospholipids, and sphingolipids in the induction of depression- and anxiety-related behaviors. These polyunsaturated fatty acids also offer new treatment options such as targeted dietary supplementation or pharmacological interference with lipid-regulating enzymes. While clinical trials support this view, effective lipid-based therapies may need more individualized approaches. Altogether, accumulating evidence suggests a crucial role for membrane lipids in the pathogenesis of depression and anxiety disorders; these lipids could be exploited for improved prevention and treatment. This article is part of a Special Issue entitled Brain Lipids.

  10. Lipid-lipid and lipid-drug interactions in biological membranes

    NASA Astrophysics Data System (ADS)

    Martynowycz, Michael W.

    Interactions between lipids and drug molecules in biological membranes help govern proper biological function in organisms. The mechanisms responsible for hydrophobic drug permeation remain elusive. Many small molecule drugs are hydrophobic. These drugs inhibit proteins in the cellular interior. The rise of antibiotic resistance in bacteria is thought to be caused by mutations in protein structure, changing drug kinetics to favor growth. However, small molecule drugs have been shown to have different mechanisms depending in the structure of the lipid membrane of the target cell. Biological membranes are investigated using Langmuir monolayers at the air-liquid interface. These offer the highest level of control in the mimetic system and allow them to be investigated using complementary techniques. Langmuir isotherms and insertion assays are used to determine the area occupied by each lipid in the membrane and the change in area caused by the introduction of a drug molecule, respectively. Specular X-ray reflectivity is used to determine the electron density of the monolayer, and grazing incidence X-ray diffraction is used to determine the in-plane order of the monolayer. These methods determine the affinity of the drug and the mechanism of action. Studies are presented on hydrophobic drugs with mammalian membrane mimics using warfarin along with modified analogues, called superwarfarins. Data shows that toxicity of these modified drugs are modulated by the membrane cholesterol content in cells; explaining several previously unexplained effects of the drugs. Membrane mimics of bacteria are investigated along with their interactions with a hydrophobic antibiotic, novobiocin. Data suggests that permeation of the drug is mediated by modifications to the membrane lipids, and completely ceases translocation under certain circumstances. Circumventing deficiencies in small, hydrophobic drugs is approached by using biologically mimetic oligomers. Peptoids, mimetic of host

  11. Electrodiffusion of lipids on membrane surfaces

    NASA Astrophysics Data System (ADS)

    Zhou, Y. C.

    2012-05-01

    Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

  12. [Germ cell membrane lipids in spermatogenesis].

    PubMed

    Wang, Ting; Shi, Xiao; Quan, Song

    2016-05-01

    Spermatogenesis is a complex developmental process in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. During spermatogenesis, membrane remodeling takes place, and cell membrane permeability and liquidity undergo phase-specific changes, which are all associated with the alteration of membrane lipids. Lipids are important components of the germ cell membrane, whose volume and ratio fluctuate in different phases of spermatogenesis. Abnormal lipid metabolism can cause spermatogenic dysfunction and consequently male infertility. Germ cell membrane lipids are mainly composed of cholesterol, phospholipids and glycolipids, which play critical roles in cell adhesion and signal transduction during spermatogenesis. An insight into the correlation of membrane lipids with spermatogenesis helps us to better understand the mechanisms of spermatogenesis and provide new approaches to the diagnosis and treatment of male infertility.

  13. Characterization of Titratable Amphiphiles in Lipid Membranes by Fluorescence Spectroscopy.

    PubMed

    Pierrat, Philippe; Lebeau, Luc

    2015-11-17

    Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent pKa value which may be as large as two pKa units and cannot be satisfactorily predicted.

  14. Finite element modeling of lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Feng, Feng; Klug, William S.

    2006-12-01

    A numerical simulation framework is presented for the study of biological membranes composed of lipid bilayers based on the finite element method. The classic model for these membranes employs a two-dimensional-fluid-like elastic constitutive law which is sensitive to curvature, and subjects vesicles to physically imposed constraints on surface area and volume. This model is implemented numerically via the use of C1-conforming triangular Loop subdivision finite elements. The validity of the framework is tested by computing equilibrium shapes from previously-determined axisymmetric shape-phase diagram of lipid bilayer vesicles with homogeneous material properties. Some of the benefits and challenges of finite element modeling of lipid bilayer systems are discussed, and it is indicated how this framework is natural for future investigation of biologically realistic bilayer structures involving nonaxisymmetric geometries, binding and adhesive interactions, heterogeneous mechanical properties, cytoskeletal interactions, and complex loading arrangements. These biologically relevant features have important consequences for the shape mechanics of nonidealized vesicles and cells, and their study requires not simply advances in theory, but also advances in numerical simulation techniques, such as those presented here.

  15. DMSO induces dehydration near lipid membrane surfaces.

    PubMed

    Cheng, Chi-Yuan; Song, Jinsuk; Pas, Jolien; Meijer, Lenny H H; Han, Songi

    2015-07-21

    Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (XDMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at XDMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At XDMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw.

  16. Accuracy of determination of position and width of molecular groups in biological and lipid membranes via neutron diffraction.

    PubMed

    Gordeliy, V I; Chernov, N I

    1997-07-01

    Neutron diffraction combined with the deuterium-labelled molecular groups of biological and model membrane components allows one to detect with high accuracy the structure of these objects. Experiments of this kind are only possible at unique high-flux neutron sources, and the planning of neutron-diffraction experiments must take into account some special requirements primarily related to the duration of the experiment and the accuracy of estimation of membrane structure parameters as a result of finite time of the measurements. This paper deals with the question of statistical accuracy of the position x(0) and width v of the distribution of deuterium labels in membranes along the normal of their plane, which are determined in a neutron diffraction experiment. It is shown that the accuracy of x(0) and v estimation does not depend on membrane constitution. It is dependent only on the scattering amplitude of the deuterium label, the label position x(0) and the distribution width v. Analytic calculations show that the statistical errors Deltax(0) and Deltav are inversely proportional to the scattering amplitude of the label and, as usual, to the square root of measurement time. The question of Deltax0 and Deltav dependence on the number of structure factors used in the calculations of x(0) and v is also studied. It is shown that, the accuracy of x(0) estimation is approximately constant with down to four structure factors used, and, with the number of the factors below four, it deteriorates drastically. Analogous is the behaviour of Deltav(h(max)) relation with one exception: abrupt deterioration of the accuracy occurs beginning with five structure factors used. One does not have to measure the highest diffraction reflections which takes a much longer time compared with the first ones. It is an important result. All the problems mentioned above have also been considered for the case of two different deuterium labels in membranes.

  17. Self-segregation of myelin membrane lipids in model membranes.

    PubMed

    Yurlova, Larisa; Kahya, Nicoletta; Aggarwal, Shweta; Kaiser, Hermann-Josef; Chiantia, Salvatore; Bakhti, Mostafa; Pewzner-Jung, Yael; Ben-David, Oshrit; Futerman, Anthony H; Brügger, Britta; Simons, Mikael

    2011-12-07

    Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.

  18. Membrane Structure: Lipid-Protein Interactions in Microsomal Membranes*

    PubMed Central

    Trump, Benjamin F.; Duttera, Sue M.; Byrne, William L.; Arstila, Antti U.

    1970-01-01

    The relationships of phospholipid to membrane structure and function were examined in hepatic microsomes. Findings indicate that normal microsomal membrane structure is dependent on lipid-protein interactions and that it correlates closely with glucose-6-phosphatase activity. Modification of most phospholipid with phospholipase-C is associated with widening of the membrane which can be reversed following readdition of phospholipid. Images PMID:4317915

  19. Biosynthesis of archaeal membrane ether lipids

    PubMed Central

    Jain, Samta; Caforio, Antonella; Driessen, Arnold J. M.

    2014-01-01

    A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria. PMID:25505460

  20. Liquid immiscibility in model bilayer lipid membranes

    NASA Astrophysics Data System (ADS)

    Veatch, Sarah L.

    There is growing evidence that cell plasma membranes are laterally organized into "raft" regions in which particular lipids and proteins are concentrated. These domains have sub-micron dimensions and have been implicated in vital cell functions. Similar liquid domains are observed in model bilayer membrane mixtures that mimick cellular lipid compositions. In model membranes, domains can be large (microns) and can readily form in the absence of proteins. This thesis presents studies of liquid immiscibility in model membrane systems using two experimental methods. By fluorescence microscopy, this thesis documents that miscibility transitions occur in a wide variety of ternary lipid mixtures containing high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol. I have constructed detailed miscibility phase diagrams for three separate ternary lipid mixtures (DOPC/DPPC/Chol, DOPC/PSM/Chol, and POPC/PSM/Chol). Phase separation is also observed in membranes of lipids extracted from human erythrocytes. NMR experiments probe lipid order and verify the coexistence of a saturated lipid and cholesterol rich liquid ordered (Lo) phase with a more disordered, unsaturated lipid rich liquid crystalline (Lalpha) phase at low temperatures. These experiments also find multiple thermodynamic transitions and lipid organization on different length-scales. This complexity is revealed because fluorescence microscopy and NMR probe lipid order at different length-scales (>1mum vs. ˜100nm). NMR detects small domains (˜80nm) at temperatures just below the miscibility transition, even though micron-scale domains are observed by fluorescent microscopy. NMR does detect large-scale ("100nm) demixing, but at a lower temperature. In addition, it has long been known that >10nm length-scale structure is present in many lipid mixtures containing cholesterol and at least one additional lipid species, though it is shown here that only a subset of

  1. Do local anesthetics interact preferentially with membrane lipid rafts? Comparative interactivities with raft-like membranes.

    PubMed

    Tsuchiya, Hironori; Ueno, Takahiro; Mizogami, Maki; Takakura, Ko

    2010-08-01

    Membranous lipid bilayers have been reconsidered as the site of action of local anesthetics (LAs). Recent understanding of biomembranes indicates the existence of lipid raft microdomains enriched in cholesterol and sphingolipids as potential platforms for channels and receptors. Based on the hypothesis that LAs may interact preferentially with lipid rafts over non-raft membranes, we compared their effects on raft model membranes and cardiolipin-containing biomimetic membranes. Liposomes were prepared with phospholipids, sphingomyelin, cerebroside, and cholesterol to have compositions corresponding to lipid rafts and cardiomyocyte mitochondrial membranes. After reacting LAs (50-200 microM) with the membrane preparations, their interactivities were determined by measuring fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene. Although bupivacaine and lidocaine acted on different raft-like liquid-ordered membranes to reduce polarization values, their effects on biomimetic less ordered membranes were much greater. LAs interacted with biomimetic membranes with the potency being R(+)-bupivacaine > racemic bupivacaine > S(-)-bupivacaine > ropivacaine > lidocaine > prilocaine, which is consistent with the rank order of pharmacotoxicological potency. However, raft model membranes showed neither structure-dependence nor stereoselectivity. The relevance of membrane lipid rafts to LAs is questionable at least in their effects on raft-like liquid-ordered membranes.

  2. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels.

    PubMed

    Glaser, Ralf W; Sachse, Carsten; Dürr, Ulrich H N; Wadhwani, Parvesh; Ulrich, Anne S

    2004-05-01

    A highly sensitive solid state (19)F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF(3)-Phg) at different positions throughout the alpha-helical peptide chain. A simple 1-pulse (19)F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF(3)-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF(3)-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF(3)-labeled peptide analogues (with L-CF(3)-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF(3)-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF(3)-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF(3)-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of

  3. Crystallizing Membrane Proteins in Lipidic Mesophases. A Host Lipid Screen

    SciTech Connect

    Li, Dianfan; Lee, Jean; Caffrey, Martin

    2011-11-30

    The default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein. There is no good reason, however, why this 18-carbon, cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins. The latter come from an array of biomembrane types with varying properties that include hydrophobic thickness, intrinsic curvature, lateral pressure profile, lipid and protein makeup, and compositional asymmetry. Thus, it seems reasonable that screening for crystallizability based on the identity of the lipid creating the hosting mesophase would be worthwhile. For this, monoacylglycerols with differing acyl chain characteristics, such as length and olefinic bond position, must be available. A lipid synthesis and purification program is in place in the author's laboratory to serve this need. In the current study with the outer membrane sugar transporter, OprB, we demonstrate the utility of host lipid screening as a means for generating diffraction-quality crystals. Host lipid screening is likely to prove a generally useful strategy for mesophase-based crystallization of membrane proteins.

  4. 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.

  5. Designing lipids for selective partitioning into liquid ordered membrane domains.

    PubMed

    Momin, Noor; Lee, Stacey; Gadok, Avinash K; Busch, David J; Bachand, George D; Hayden, Carl C; Stachowiak, Jeanne C; Sasaki, Darryl Y

    2015-04-28

    Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

  6. Lipids and topological rules governing membrane protein assembly☆

    PubMed Central

    Bogdanov, Mikhail; Dowhan, William; Vitrac, Heidi

    2014-01-01

    Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein Trafficking

  7. Unconventional membrane lipid biosynthesis in Xanthomonas campestris.

    PubMed

    Aktas, Meriyem; Narberhaus, Franz

    2015-09-01

    All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) is well understood in model bacteria such as Escherichia coli. It recently emerged, however, that the diversity of bacterial membrane lipids is huge and that not yet explored biosynthesis pathways exist, even for the common PLs. A good example is the plant pathogen Xanthomonas campestris pv. campestris. It contains PE, PG and CL as major lipids and small amounts of the N-methylated PE derivatives monomethyl PE and phosphatidylcholine (PC = trimethylated PE). Xanthomonas campestris uses a repertoire of canonical and non-canonical enzymes for the synthesis of its membrane lipids. In this minireview, we briefly recapitulate standard pathways and integrate three recently discovered pathways into the overall picture of bacterial membrane biosynthesis.

  8. 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

  9. [Role of membrane lipids in myocardial cytoprotection

    NASA Technical Reports Server (NTRS)

    Grynberg, A.

    2000-01-01

    The cardiomyocyte capacity to regulate ATP production to face any change in energy demand is a major determinant of cardiac function. This process is based on a balanced fatty acid (FA) metabolism, because FA is the main fuel of the heart, although the most expensive one in oxygen. The pathway is, however, weakly controlled by the cardiac myocyte which can well regulate FA mitochondrial entry but not cell FA uptake. For this reason, several pathological situations often result from either harmful accumulation of FA and derivatives or excess FA-oxidation. Control of the FA/glucose balance by decreased energy production from FA would thus offer an alternative strategy in the treatment of ischaemia, providing the cardiomyocytes weak ability in handling the non-metabolised FA is controlled. The initiation and the regulation of cardiac contraction both result from membrane activity; the other major role of lipids in the heart is their contribution to membrane homeostasis through phospholipid synthesis pathways and phospholipases. The anti-anginal activity of Trimetazidine, reported as a cytoprotective effect without a haemo-dynamic component; is associated with reduced use of FA for energy. However, accumulation of FA and derivatives has never been observed. Trimetazidine is reported to increase significantly the synthesis of phospholipids without influencing the other lipid classes, thus increasing the incorporation of FA in membrane structures. This cytoprotection appears to be based on the redirection of the use of FA to phospholipid synthesis, which would decrease their availability for energy production. This class of compounds, with the same properties as Trimetazidine, offers a metabolic approach to the treatment of ischaemia.

  10. Effect of Different Broad Waveband Lights on Membrane Lipids of a Cyanobacterium, Synechococcus sp., as Determined by UPLC-QToF-MS and Vibrational Spectroscopy.

    PubMed

    Montero, Olimpio; Velasco, Marta; Sanz-Arranz, Aurelio; Rull, Fernando

    2016-05-23

    Differential profile of membrane lipids and pigments of a Synechococcus sp. cyanobacterial strain cells exposed to blue, green, red and white light are determined by means of liquid chromatography and mass spectrometry or diode array detection. Raman and ATR-IR spectra of intact cells under the diverse light wavebands are also reported. Blue light cells exhibited an increased content of photosynthetic pigments as well as specific species of membrane glycerolipids as compared to cells exposed to other wavebands. The A630/A680 ratio indicated an increased content of phycobilisomes (PBS) in the blue light-exposed cells. Some differences in the protein conformation between the four light waveband-exposed cells were deduced from the variable absorbance at specific wavenumbers in the FT-Raman and ATR-FTIR spectra, in particular bands assigned to amide I and amide II. Bands from 1180 to 950 cm(-1) in the ATR-FTIR spectrum suggest degraded outer membrane polysaccharide in the blue light-exposed cells.

  11. Effect of Different Broad Waveband Lights on Membrane Lipids of a Cyanobacterium, Synechococcus sp., as Determined by UPLC-QToF-MS and Vibrational Spectroscopy

    PubMed Central

    Montero, Olimpio; Velasco, Marta; Sanz-Arranz, Aurelio; Rull, Fernando

    2016-01-01

    Differential profile of membrane lipids and pigments of a Synechococcus sp. cyanobacterial strain cells exposed to blue, green, red and white light are determined by means of liquid chromatography and mass spectrometry or diode array detection. Raman and ATR-IR spectra of intact cells under the diverse light wavebands are also reported. Blue light cells exhibited an increased content of photosynthetic pigments as well as specific species of membrane glycerolipids as compared to cells exposed to other wavebands. The A630/A680 ratio indicated an increased content of phycobilisomes (PBS) in the blue light-exposed cells. Some differences in the protein conformation between the four light waveband-exposed cells were deduced from the variable absorbance at specific wavenumbers in the FT-Raman and ATR-FTIR spectra, in particular bands assigned to amide I and amide II. Bands from 1180 to 950 cm−1 in the ATR-FTIR spectrum suggest degraded outer membrane polysaccharide in the blue light-exposed cells. PMID:27223306

  12. Pore dynamics in lipid membranes

    NASA Astrophysics Data System (ADS)

    Gozen, I.; Dommersnes, P.

    2014-09-01

    Transient circular pores can open in plasma membrane of cells due to mechanical stress, and failure to repair such pores lead to cell death. Similar pores in the form of defects also exist among smectic membranes, such as in myelin sheaths or mitochondrial membranes. The formation and growth of membrane defects are associated with diseases, for example multiple sclerosis. A deeper understanding of membrane pore dynamics can provide a more refined picture of membrane integrity-related disease development, and possibly also treatment options and strategies. Pore dynamics is also of great importance regarding healthcare applications such as drug delivery, gene or as recently been implied, cancer therapy. The dynamics of pores significantly differ in stacks which are confined in 2D compared to those in cells or vesicles. In this short review, we will summarize the dynamics of different types of pores that can be observed in biological membranes, which include circular transient, fusion and hemi-fusion pores. We will dedicate a section to floral and fractal pores which were discovered a few years ago and have highly peculiar characteristics. Finally, we will discuss the repair mechanisms of large area pores in conjunction with the current cell membrane repair hypotheses.

  13. Steady-state compartmentalization of lipid membranes by active proteins.

    PubMed Central

    Sabra, M C; Mouritsen, O G

    1998-01-01

    Using a simple microscopic model of lipid-protein interactions, based on the hydrophobic matching principle, we study some generic aspects of lipid-membrane compartmentalization controlled by a dispersion of active integral membrane proteins. The activity of the proteins is simulated by conformational excitations governed by an external drive, and the deexcitation is controlled by interaction of the protein with its lipid surroundings. In response to the flux of energy into the proteins from the environment and the subsequent dissipation of energy into the lipid bilayer, the lipid-protein assembly reorganizes into a steady-state structure with a typical length scale determined by the strength of the external drive. In the specific case of a mixed dimyristoylphosphatidylcholine-distearoylphosphatidylcholine bilayer in the gel-fluid coexistence region, it is shown explicitly by computer simulation that the activity of an integral membrane protein can lead to a compartmentalization of the lipid-bilayer membrane. The compartmentalization is related to the dynamical process of phase separation and lipid domain formation. PMID:9533687

  14. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.

    PubMed

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-10-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.

  15. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer

    PubMed Central

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-01-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. PMID:26269359

  16. Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

    PubMed Central

    Reddy, A. Srinivas; Warshaviak, Dora Toledo; Chachisvilis, Mirianas

    2013-01-01

    Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15 dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ∼5% and ∼5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S2) and deuterium (SCD) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10– 20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15 dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells. PMID:22588133

  17. Nanosecond Lipid Dynamics in Membranes Containing Cholesterol

    SciTech Connect

    Armstrong, Clare L; Haeussler, Wolfgang; Seydel, Tilo; Katsaras, John; Rheinstadter, Maikel C

    2014-01-01

    Lipid dynamics in the cholesterol-rich (40 mol%) liquid-ordered (lo) phase of dimyristoylphosphatidylcholine membranes were studied using neutron spin-echo and neutron backscattering. Recent theoretical and experimental evidence supports the notion of the liquid-ordered phase in phospholipid membranes as a locally structured liquid, with small ordered domains of a highly dynamic nature in equilibrium with a disordered matrix [S. Meinhardt, R. L. C. Vink and F. Schmid, Proc. Natl. Acad. Sci. U. S. A., 2013, 110(12), 4476 4481, C. L. Armstrong et al., PLoS One, 2013, 8(6), e66162]. This local structure was found to have a pronounced impact on the membranes' dynamical properties. We found that the long-wavelength dynamics in the liquid-ordered phase, associated with the elastic properties of the membranes, were faster by two orders of magnitude as compared to the liquid disordered phase. At the same time, collective nanoscale diffusion was significantly slower. The presence of a soft-mode (a slowing down) in the longwavelength dispersion relationship suggests an upper size limit for the ordered lipid domain of ~220 A. Moreover, from the relaxation rate of the collective lipid diffusion of lipid lipid distances, the lifetime of these domains was estimated to be about 100 nanoseconds.

  18. Preparation of supported lipid membranes for aquaporin Z incorporation.

    PubMed

    Li, Xuesong; Wang, Rong; Tang, Chuyang; Vararattanavech, Ardcharaporn; Zhao, Yang; Torres, Jaume; Fane, Tony

    2012-06-01

    There has been a recent surge of interest to mimic the performance of natural cellular membranes by incorporating water channel proteins-aquaporins (AQPs) into various ultrathin films for water filtration applications. To make biomimetic membranes one of the most crucial steps is preparing a defect-free platform for AQPs incorporation on a suitable substrate. In this study two methods were used to prepare supported lipid membranes on NF membrane surfaces under a benign pH condition of 7.8. One method was direct vesicle fusion on a hydrophilic membrane NF-270; the other was vesicle fusion facilitated by hydraulic pressure on a modified hydrophilic NF-270 membrane whose surface has been spin-coated with positively charged lipids. Experiments revealed that the supported lipid membrane without AQPs prepared by the spin coating plus vesicle fusion had a much lower defect density than that prepared by vesicle fusion alone. It appears that the surface roughness and charge are the main factors determining the quality of the supported lipid membrane. Aquaporin Z (AqpZ) proteins were successfully incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes and its permeability was measured by the stopped-flow experimental procedure. However, after the proteoliposomes have been fused onto the modified substrate, the AqpZ function in the resultant membrane was not observed and AFM images showed distinct aggregations of unfused proteoliposomes or AqpZ proteins on the substrate surface. It is speculated that the inhibition of AqpZ function may be caused by the low lipid mobility on the NF membrane surface. Further investigations to evaluate and optimize the structure-performance relationship are required.

  19. Critical point fluctuations in supported lipid membranes.

    PubMed

    Connell, Simon D; Heath, George; Olmsted, Peter D; Kisil, Anastasia

    2013-01-01

    In this paper, we demonstrate that it is possible to observe many aspects of critical phenomena in supported lipid bilayers using atomic force microscopy (AFM) with the aid of stable and precise temperature control. The regions of criticality were determined by accurately measuring and calculating phase diagrams for the 2 phase L(d)-L(o) region, and tracking how it moves with temperature, then increasing the sampling density around the estimated critical regions. Compositional fluctuations were observed above the critical temperature (T(c)) and characterised using a spatial correlation function. From this analysis, the phase transition was found to be most closely described by the 2D Ising model, showing it is a critical transition. Below T(c) roughening of the domain boundaries occurred due to the reduction in line tension close to the critical point. Smaller scale density fluctuations were also detected just below T(c). At T(c), we believe we have observed fluctuations on length scales greater than 10 microm. The region of critically fluctuating 10-100 nm nanodomains has been found to extend a considerable distance above T(c) to temperatures within the biological range, and seem to be an ideal candidate for the actual structure of lipid rafts in cell membranes. Although evidence for this idea has recently emerged, this is the first direct evidence for nanoscale domains in the critical region.

  20. 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

  1. TOPICAL REVIEW: The microcalorimetry of lipid membranes

    NASA Astrophysics Data System (ADS)

    Heerklotz, Heiko

    2004-04-01

    Insight into the forces governing a system is essential for understanding its behaviour and function. Calorimetric investigations provide a wealth of information that is not, or is hardly, available by other methods. This paper reviews calorimetric approaches and assays for the study of lipid vesicles (liposomes) and biological membranes. With respect to the instrumentation, differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), isothermal titration calorimetry (ITC) and water sorption calorimetry are considered. Applications of these techniques to lipid systems include the measurement of thermodynamic parameters and a detailed characterization of the thermotropic, barotropic, and lyotropic phase behaviour. The membrane binding or partitioning of solutes (proteins, peptides, drugs, surfactants, ions, etc) can also be quantified. Many calorimetric assays are available for studying the effect of proteins and other additives on membranes, characterizing non-ideal mixing, domain formation, stability, curvature strain, permeability, solubilization, and fusion. Studies of membrane proteins in lipid environments elucidate lipid-protein interactions in membranes. The systems are described in terms of enthalpic and entropic forces, equilibrium constants, heat capacities, partial volume changes etc, shedding light also on the stability of structures and the molecular origin and mechanism of structural changes.

  2. Nonlinear adhesion dynamics of confined lipid membranes

    NASA Astrophysics Data System (ADS)

    To, Tung; Le Goff, Thomas; Pierre-Louis, Olivier

    Lipid membranes, which are ubiquitous objects in biological environments are often confined. For example, they can be sandwiched between a substrate and the cytoskeleton between cell adhesion, or between other membranes in stacks, or in the Golgi apparatus. We present a study of the nonlinear dynamics of membranes in a model system, where the membrane is confined between two flat walls. The dynamics derived from the lubrication approximation is highly nonlinear and nonlocal. The solution of this model in one dimension exhibits frozen states due to oscillatory interactions between membranes caused by the bending rigidity. We develope a kink model for these phenomena based on the historical work of Kawasaki and Otha. In two dimensions, the dynamics is more complex, and depends strongly on the amount of excess area in the system. We discuss the relevance of our findings for experiments on model membranes, and for biological systems. Supported by the grand ANR Biolub.

  3. Stearoyl-CoA Desaturase 1 Is a Key Determinant of Membrane Lipid Composition in 3T3-L1 Adipocytes

    PubMed Central

    Hagen, Rachel; Vidal-Puig, Antonio

    2016-01-01

    Stearoyl-CoA desaturase 1 (SCD1) is a lipogenic enzyme important for the regulation of membrane lipid homeostasis; dysregulation likely contributes to obesity associated metabolic disturbances. SCD1 catalyses the Δ9 desaturation of 12-19 carbon saturated fatty acids to monounsaturated fatty acids. To understand its influence in cellular lipid composition we investigated the effect of genetic ablation of SCD1 in 3T3-L1 adipocytes on membrane microdomain lipid composition at the species-specific level. Using liquid chromatography/electrospray ionisation-tandem mass spectrometry, we quantified 70 species of ceramide, mono-, di- and trihexosylceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, bis(monoacylglycero)phosphate, phosphatidylinositol and cholesterol in 3T3-L1 adipocytes in which a 90% reduction in scd1 mRNA expression was achieved with siRNA. Cholesterol content was unchanged although decreases in other lipids resulted in cholesterol accounting for a higher proportion of lipid in the membranes. This was associated with decreased membrane lateral diffusion. An increased ratio of 24:0 to 24:1 in ceramide, mono- and dihexosylceramide, and sphingomyelin likely also contributed to this decrease in lateral diffusion. Of particular interest, we observed a decrease in phospholipids containing arachidonic acid. Given the high degree of structural flexibility of this acyl chain this will influence membrane lateral diffusion, and is likely responsible for the transcriptional activation of Lands’ cycle enzymes lpcat3 and mboat7. Of relevance these profound changes in the lipidome were not accompanied by dramatic changes in gene expression in mature differentiated adipocytes, suggesting that adaptive homeostatic mechanisms to ensure partial maintenance of the biophysical properties of membranes likely occur at a post-transcriptional level. PMID:27632198

  4. Bacterial membrane lipids: diversity in structures and pathways.

    PubMed

    Sohlenkamp, Christian; Geiger, Otto

    2016-01-01

    For many decades, Escherichia coli was the main model organism for the study of bacterial membrane lipids. The results obtained served as a blueprint for membrane lipid biochemistry, but it is clear now that there is no such thing as a typical bacterial membrane lipid composition. Different bacterial species display different membrane compositions and even the membrane composition of cells belonging to a single species is not constant, but depends on the environmental conditions to which the cells are exposed. Bacterial membranes present a large diversity of amphiphilic lipids, including the common phospholipids phosphatidylglycerol, phosphatidylethanolamine and cardiolipin, the less frequent phospholipids phosphatidylcholine, and phosphatidylinositol and a variety of other membrane lipids, such as for example ornithine lipids, glycolipids, sphingolipids or hopanoids among others. In this review, we give an overview about the membrane lipid structures known in bacteria, the different metabolic pathways involved in their formation, and the distribution of membrane lipids and metabolic pathways across taxonomical groups.

  5. Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing

    PubMed Central

    Sezgin, Erdinc; Waithe, Dominic; Bernardino de la Serna, Jorge; Eggeling, Christian

    2015-01-01

    Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes. PMID:25755090

  6. Atomistic Monte Carlo Simulation of Lipid Membranes

    PubMed Central

    Wüstner, Daniel; Sklenar, Heinz

    2014-01-01

    Biological membranes are complex assemblies of many different molecules of which analysis demands a variety of experimental and computational approaches. In this article, we explain challenges and advantages of atomistic Monte Carlo (MC) simulation of lipid membranes. We provide an introduction into the various move sets that are implemented in current MC methods for efficient conformational sampling of lipids and other molecules. In the second part, we demonstrate for a concrete example, how an atomistic local-move set can be implemented for MC simulations of phospholipid monomers and bilayer patches. We use our recently devised chain breakage/closure (CBC) local move set in the bond-/torsion angle space with the constant-bond-length approximation (CBLA) for the phospholipid dipalmitoylphosphatidylcholine (DPPC). We demonstrate rapid conformational equilibration for a single DPPC molecule, as assessed by calculation of molecular energies and entropies. We also show transition from a crystalline-like to a fluid DPPC bilayer by the CBC local-move MC method, as indicated by the electron density profile, head group orientation, area per lipid, and whole-lipid displacements. We discuss the potential of local-move MC methods in combination with molecular dynamics simulations, for example, for studying multi-component lipid membranes containing cholesterol. PMID:24469314

  7. Theoretical and simulation study of lipid membranes

    NASA Astrophysics Data System (ADS)

    Khelashvili, George

    It has been established that a proper functioning of biological lipid membranes is in large part due to cholesterol's ability to regulate fluidity of a lipid bilayer. In particular, a growing body of evidence suggested that cholesterol participates in the formation of cholesterol- and sphingolipid-enriched phase-separated domains known as "rafts" in the plasma and other membranes of animal cells. Rafts have been identified as important membrane structural components in signal transduction, protein transport and sorting of membrane components. At a molecular level, the detailed, localized behavior of lipid-cholesterol bilayers is unclear. In order to better understand how cholesterols function in lipid membranes it is desirable to built theoretical models. The goal of the present research is to model lipid-cholesterol bilayers on the different length and timescales. In the first part of the work, mixtures of sphingomyelin (SM) lipid and cholesterol at different temperatures and cholesterol concentrations were investigated using Molecular Dynamics and Monte-Carlo simulation techniques. The objective was to study the properties of cholesterol- and SM-enriched raft-like domains at the atomic level. The simulations revealed that, addition of 31% cholesterol induced intermediate degree of organization in the model SM-cholesterol bilayers at temperatures below and above the main phase transition temperature of pure SM bilayer. This intermediate state of fluidity may be necessary for the binding of proteins and other molecules that associate with raft domains. In the second part of the work, dynamical self-consistent mean-field model based on atomistic simulations was developed to investigate phase properties of lipid-cholesterol bilayers on the length and timescales currently unreachable with traditional atomistic level simulation methods. This new technique allows studying systems consisting of 104 or more number of molecules, on microsecond timescales. The model was

  8. The use of cis-parinaric acid to determine lipid peroxidation in human erythrocyte membranes. Comparison of normal and sickle erythrocyte membranes.

    PubMed

    Van den Berg, J J; Kuypers, F A; Qju, J H; Chiu, D; Lubin, B; Roelofsen, B; Op den Kamp, J A

    1988-09-15

    The recently developed parinaric acid assay is shown to offer possibilities for studying peroxidation processes in biological membrane systems. Taking the human erythrocyte membrane as a model, several initiating systems were investigated, as well as the effect of residual hemoglobin in ghost membrane preparations. The effectivity of a radical generating system appeared to be strongly dependent upon whether radicals are generated at the membrane level or in the water phase. Thus, cumene hydroperoxide at concentrations of 1.0-1.5 mM was found to be a very efficient initiator of peroxidation in combination with submicromolar levels of hemin-Fe3+ as membrane-bound cofactor. In combination with cumene hydroperoxide, membrane-bound hemoglobin appeared to be about 6-times more effective in promoting peroxidation than hemoglobin in the water phase. Results comparing the behaviour of normal and sickle erythrocyte ghost suspensions in the peroxidation assay suggest that the increased oxidative stress on sickle erythrocyte membranes could be due to enhanced membrane binding of sickle hemoglobin, but also partly to a characteristically higher capability of sickle hemoglobin to promote peroxidation. The order of peroxidation-promoting capabilities that could be derived from the experiments was hemin greater than sickle hemoglobin greater than normal hemoglobin.

  9. Kinetic and thermodynamic aspects of lipid translocation in biological membranes.

    PubMed Central

    Frickenhaus, S; Heinrich, R

    1999-01-01

    A theoretical analysis of the lipid translocation in cellular bilayer membranes is presented. We focus on an integrative model of active and passive transport processes determining the asymmetrical distribution of the major lipid components between the monolayers. The active translocation of the aminophospholipids phosphatidylserine and phosphatidylethanolamine is mathematically described by kinetic equations resulting from a realistic ATP-dependent transport mechanism. Concerning the passive transport of the aminophospholipids as well as of phosphatidylcholine, sphingomyelin, and cholesterol, two different approaches are used. The first treatment makes use of thermodynamic flux-force relationships. Relevant forces are transversal concentration differences of the lipids as well as differences in the mechanical states of the monolayers due to lateral compressions. Both forces, originating primarily from the operation of an aminophospholipid translocase, are expressed as functions of the lipid compositions of the two monolayers. In the case of mechanical forces, lipid-specific parameters such as different molecular surface areas and compression force constants are taken into account. Using invariance principles, it is shown how the phenomenological coefficients depend on the total lipid amounts. In a second approach, passive transport is analyzed in terms of kinetic mechanisms of carrier-mediated translocation, where mechanical effects are incorporated into the translocation rate constants. The thermodynamic as well as the kinetic approach are applied to simulate the time-dependent redistribution of the lipid components in human red blood cells. In the thermodynamic model the steady-state asymmetrical lipid distribution of erythrocyte membranes is simulated well under certain parameter restrictions: 1) the time scales of uncoupled passive transbilayer movement must be different among the lipid species; 2) positive cross-couplings of the passive lipid fluxes are

  10. Targeting Membrane Lipid a Potential Cancer Cure?

    PubMed Central

    Tan, Loh Teng-Hern; Chan, Kok-Gan; Pusparajah, Priyia; Lee, Wai-Leng; Chuah, Lay-Hong; Khan, Tahir Mehmood; Lee, Learn-Han; Goh, Bey-Hing

    2017-01-01

    Cancer mortality and morbidity is projected to increase significantly over the next few decades. Current chemotherapeutic strategies have significant limitations, and there is great interest in seeking novel therapies which are capable of specifically targeting cancer cells. Given that fundamental differences exist between the cellular membranes of healthy cells and tumor cells, novel therapies based on targeting membrane lipids in cancer cells is a promising approach that deserves attention in the field of anticancer drug development. Phosphatidylethanolamine (PE), a lipid membrane component which exists only in the inner leaflet of cell membrane under normal circumstances, has increased surface representation on the outer membrane of tumor cells with disrupted membrane asymmetry. PE thus represents a potential chemotherapeutic target as the higher exposure of PE on the membrane surface of cancer cells. This feature as well as a high degree of expression of PE on endothelial cells in tumor vasculature, makes PE an attractive molecular target for future cancer interventions. There have already been several small molecules and membrane-active peptides identified which bind specifically to the PE molecules on the cancer cell membrane, subsequently inducing membrane disruption leading to cell lysis. This approach opens up a new front in the battle against cancer, and is of particular interest as it may be a strategy that may be prove effective against tumors that respond poorly to current chemotherapeutic agents. We aim to highlight the evidence suggesting that PE is a strong candidate to be explored as a potential molecular target for membrane targeted novel anticancer therapy. PMID:28167913

  11. Determination of uptake kinetics (sampling rates) by lipid-containing semipermeable membrane devices (SPMDs) for polycyclic aromatic hydrocarbons (PAHs) in water

    USGS Publications Warehouse

    Huckins, J.N.; Petty, J.D.; Orazio, C.E.; Lebo, J.A.; Clark, R.C.; Gibson, V.L.; Gala, W.R.; Echols, K.R.

    1999-01-01

    The use of lipid-containing semipermeable membrane devices (SPMDs) is becoming commonplace, but very little sampling rate data are available for the estimation of ambient contaminant concentrations from analyte levels in exposed SPMDs. We determined the aqueous sampling rates (R(s)s; expressed as effective volumes of water extracted daily) of the standard (commercially available design) 1-g triolein SPMD for 15 of the priority pollutant (PP) polycyclic aromatic hydrocarbons (PAHs) at multiple temperatures and concentrations. Under the experimental conditions of this study, recovery- corrected R(s) values for PP PAHs ranged from ???1.0 to 8.0 L/d. These values would be expected to be influenced by significant changes (relative to this study) in water temperature, degree of biofouling, and current velocity- turbulence. Included in this paper is a discussion of the effects of temperature and octanol-water partition coefficient (K(ow)); the impacts of biofouling and hydrodynamics are reported separately. Overall, SPMDs responded proportionally to aqueous PAH concentrations; i.e., SPMD R(s) values and SPMD-water concentration factors were independent of aqueous concentrations. Temperature effects (10, 18, and 26 ??C) on Rs values appeared to be complex but were relatively small.The use of lipid-containing semipermeable membrane devices (SPMDs) is becoming commonplace, but very little sampling rate data are available for the estimation of ambient contaminant concentrations from analyte levels in exposed SPMDs. We determined the aqueous sampling rates (Rss; expressed as effective volumes of water extracted daily) of the standard (commercially available design) 1-g triolein SPMD for 15 of the priority pollutant (PP) polycyclic aromatic hydrocarbons (PAHs) at multiple temperatures and concentrations. Under the experimental conditions of this study, recovery-corrected Rs values for PP PAHs ranged from ???1.0 to 8.0 L/d. These values would be expected to be influenced by

  12. Self-assembled tethered bimolecular lipid membranes.

    PubMed

    Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

    2009-01-01

    This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules

  13. Anionic lipids and the maintenance of membrane electrostatics in eukaryotes

    PubMed Central

    Platre, Matthieu Pierre

    2017-01-01

    ABSTRACT A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells. PMID:28102755

  14. Determination of uptake kinetics (sampling rates) by lipid-containing semipermeable membrane devices (SPMDs) for polycyclic aromatic hydrocarbons (PAHs) in water

    SciTech Connect

    Huckins, J.N.; Petty, J.D.; Orazio, C.E.; Lebo, J.A.; Clark, R.C.; Gibson, V.L.; Gala, W.R.; Echols, K.R.

    1999-11-01

    The use of lipid-containing semipermeable membrane devices (SPMDs) is becoming commonplace, but very little sampling rate data are available for the estimation of ambient contaminant concentrations from analyze levels in exposed SPMDs. The authors determined the aqueous sampling rates (R{sub s}s; expressed as effective volumes of water extracted daily) of the standard (commercially available design) 1-g triolein SPMD for 15 of the priority pollutant (PP) polycyclic aromatic hydrocarbons (PAHs) at multiple temperatures and concentrations. Under the experimental conditions of this study, recovery-corrected R{sub s} values for PP PAHs ranged from {approximately}1.0 to 8.0 L/d. These values would be expected to be influenced by significant changes (relative to this study) in water temperature, degree of biofouling, and current velocity-turbulence. Included in this paper is a discussion of the effects of temperature and octanol-water partition coefficient (K{sub ow}); the impacts of biofouling and hydrodynamics are reported separately. Overall, SPMDs responded proportionally to aqueous PAH concentrations; i.e., SPMD R{sub s} values and SPMD-water concentration factors were independent of aqueous concentrations. Temperature effects on R{sub s} values appeared to be complex but were relatively small.

  15. Membrane lipids and the origin of life

    NASA Technical Reports Server (NTRS)

    Oro, J.; Holzer, G.; Rao, M.; Tornabene, T. G.

    1981-01-01

    The current state of knowledge regarding the development of biological systems is briefly reviewed. At a crucial stage concerning the evolution of such systems, the mechanisms leading to more complex structures must have evolved within the confines of a protected microenvironment, similar to those provided by the contemporary cell membranes. The major components found normally in biomembranes are phospholipids. The structure of the biomembrane is examined, and attention is given to questions concerning the availability of the structural components which are necessary in the formation of primitive lipid membranes. Two approaches regarding the study of protomembranes are discussed. The probability of obtaining ether lipids under prebiotic conditions is considered, taking into account the formation of cyclic and acyclic isoprenoids by the irradiation of isoprene with UV.

  16. Pore formation in lipid membranes by alamethicin.

    PubMed Central

    Fringeli, U P; Fringeli, M

    1979-01-01

    The conformation of the linear peptide antibiotic alamethicin in dipalmitoyl phosphatidylcholine multilayers was investigated in the absence of an electric field by means of infrared attenuated total reflection spectroscopy. Alamethicin was found to be incorporated into the lipid membrane not only in the dry state but also in an aqueous environment. Its molecular conformation, however, changed from a helix when dry to an extended chain when aqueous. The extended chain aggregated to di- and multimers spanning the lipid bilayer. The equilibrium concentration of alamethicin in the surrounding water was 90 nM, which is in the range of concentrations used in black film experiments. The corresponding molar ratio of lipid to peptide was 80:1. Concerning the molecular mechanism of electric field-induced pore formation, one has to conclude that the dipole model proposed by several authors is very unlikely because it is based on the assumption that the major part of alamethicin is adsorbed on the membrane surface, from which small amounts flip into the membrane under the influence of an electric field. An alternative mechanism is proposed, based on a field-induced conformational change of the peptide from the extended state to a helix. This transition is favored by the resulting dipole moment of the alamethicin helix. PMID:291045

  17. Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy.

    PubMed

    Matam, Yashasvi; Ray, Bruce D; Petrache, Horia I

    2016-04-08

    Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain's reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells.

  18. Multichannel taste sensors with lipid, lipid like polymer membranes

    NASA Astrophysics Data System (ADS)

    Szpakowska, M.; Szwacki, J.; Marjańska, E.

    2008-08-01

    The elaboration of a sensitive taste sensor for discrimination of different soft drinks is very important in food industry. The short review of taste sensors described in the literature is presented. Two types of potentiometric taste sensors, one with lipophilic compound-polymer membranes (ISE) and the other with lipid polymer membrane and a conducting polymer film (All solid state electrode, ASSE) were tested in appropriate taste solutions. Five channel ISE sensor was examined in acid, sour and sweet solutions. This sensor was sensitive to bitter and sour substances and not too sensitive to sucrose concentration. It was successfully used for discrimination of different kind of soft drinks. Four channel ASSE sensor was examined in sour solutions. It was found that stability and sensitivity of ASSE are lower than ISE. Therefore, it seems that the previous one cannot be applied in taste sensor.

  19. Ethanol production and maximum cell growth are highly correlated with membrane lipid composition during fermentation as determined by lipidomic analysis of 22 Saccharomyces cerevisiae strains.

    PubMed

    Henderson, Clark M; Lozada-Contreras, Michelle; Jiranek, Vladimir; Longo, Marjorie L; Block, David E

    2013-01-01

    Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R(2) = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R(2) = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.

  20. Lipid Gymnastics: Tethers and Fingers in membrane

    NASA Astrophysics Data System (ADS)

    Tayebi, Lobat; Miller, Gregory; Parikh, Atul

    2009-03-01

    A significant body of evidence now links local mesoscopic structure (e.g., shape and composition) of the cell membrane with its function; the mechanisms by which cellular membranes adopt the specific shapes remain poorly understood. Among all the different structures adopted by cellular membranes, the tubular shape is one of the most surprising one. While their formation is typically attributed to the reorganization of membrane cytoskeleton, many exceptions exist. We report the instantaneous formation of tubular membrane mesophases following the hydration under specific thermal conditions. The shapes emerge in a bimodal way where we have two distinct diameter ranges for tubes, ˜20μm and ˜1μm, namely fat fingers and narrow tethers. We study the roughening of hydrated drops of 3 lipids in 3 different spontaneous curvatures at various temp. and ionic strength to figure out the dominant effect in selection of tethers and fingers. Dynamics of the tubes are of particular interest where we observe four distinct steps of birth, coiling, uncoiling and retraction with different lifetime on different thermal condition. These dynamics appear to reflect interplay between membrane elasticity, surface adhesion, and thermal or hydrodynamic gradient.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  2. Three-Phase Coexistence in Lipid Membranes.

    PubMed

    Aufderhorst-Roberts, Anders; Chandra, Udayan; Connell, Simon D

    2017-01-24

    Phospholipid ternary systems are useful model systems for understanding lipid-lipid interactions and their influence on biological properties such as cell signaling and protein translocation. Despite extensive studies, there are still open questions relating to membrane phase behavior, particularly relating to a proposed state of three-phase coexistence, due to the difficulty in clearly distinguishing the three phases. We look in and around the region of the phase diagram where three phases are expected and use a combination of different atomic force microscopy (AFM) modes to present the first images of three coexisting lipid phases in biomimetic cell lipid membranes. Domains form through either nucleation or spinodal decomposition dependent upon composition, with some exhibiting both mechanisms in different domains simultaneously. Slow cooling rates are necessary to sufficiently separate mixtures with high proportions of lo and lβ phase. We probe domain heights and mechanical properties and demonstrate that the gel (lβ) domains have unusually low structural integrity in the three-phase region. This finding supports the hypothesis of a "disordered gel" state that has been proposed from NMR studies of similar systems, where the addition of small amounts of cholesterol was shown to disrupt the regular packing of the lβ state. We use NMR data from the literature on chain disorder in different mixtures and estimate an expected step height that is in excellent agreement with the AFM data. Alternatively, the disordered solid phase observed here and in the wider literature could be explained by the lβ phase being out of equilibrium, in a surface kinetically trapped state. This view is supported by the observation of unusual growth of nucleated domains, which we term "tree-ring growth," reflecting compositional heterogeneity in large disordered lβ phase domains.

  3. Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

    PubMed

    Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

    2014-01-22

    Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.

  4. A sliding selectivity scale for lipid binding to membrane proteins

    PubMed Central

    Landreh, Michael; Marty, Michael T.; Gault, Joseph; Robinson, Carol V.

    2017-01-01

    Biological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins that reside in the membrane have co-evolved with their hydrophobic lipid environment which serves as a solvent for proteins with very diverse requirements. As a result, membrane protein-lipid interactions range from completely non-selective to highly discriminating. Mass spectrometry (MS), in combination with X-ray crystallography and molecular dynamics simulations, enables us to monitor how lipids interact with intact membrane protein complexes and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. In this review, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization. PMID:27155089

  5. Force Field Development for Lipid Membrane Simulations.

    PubMed

    Lyubartsev, Alexander P; Rabinovich, Alexander L

    2016-10-01

    With the rapid development of computer power and wide availability of modelling software computer simulations of realistic models of lipid membranes, including their interactions with various molecular species, polypeptides and membrane proteins have become feasible for many research groups. The crucial issue of the reliability of such simulations is the quality of the force field, and many efforts, especially in the latest several years, have been devoted to parametrization and optimization of the force fields for biomembrane modelling. In this review, we give account of the recent development in this area, covering different classes of force fields, principles of the force field parametrization, comparison of the force fields, and their experimental validation. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

  6. Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

    PubMed Central

    Quon, Evan; Beh, Christopher T.

    2015-01-01

    Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

  7. Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

    PubMed Central

    Shi, Liang; Jiang, Qiu-Xing

    2013-01-01

    To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels. PMID:23892292

  8. Studying lipid organization in biological membranes using liposomes and EPR spin labeling

    PubMed Central

    Subczynski, Witold K.; Raguz, Marija; Widomska, Justyna

    2015-01-01

    Summary Electron paramagnetic resonance (EPR) spin-labeling methods provide a unique opportunity to determine the lateral organization of lipid bilayer membranes by discrimination of coexisting membrane domains or coexisting membrane phases. In some cases, the coexisting membrane domains can be characterized by profiles of alkyl chain order, fluidity, hydrophobicity, and oxygen diffusion-concentration product in situ, without the need for their physical separation. This chapter briefly explains how the EPR spin-labeling methods can be used to obtain the above mentioned profiles across the lipid bilayer membranes (liposomes) derived from the lipid extract of certain biological membranes. These procedures will be illustrated by EPR measurements performed for multilamellar liposomes made of the lipid extracts from cortical and nuclear fractions of the fiber cell plasma membranes of a cow eye lens. To elucidate better the major factors that determine membrane properties, the results for eye lens lipid membranes will be compared with those obtained for simple model membranes resembling basic lipid composition of biological membranes. PMID:20013402

  9. Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry.

    PubMed

    Lewis, Kenneth T; Maddipati, Krishna R; Naik, Akshata R; Jena, Bhanu P

    2017-03-02

    Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.

  10. Determination of molecular groups involved in the interaction of annexin A5 with lipid membrane models at the air-water interface.

    PubMed

    Fezoua-Boubegtiten, Zahia; Desbat, Bernard; Brisson, Alain; Lecomte, Sophie

    2010-06-01

    Annexin A5 (AnxA5) is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. In this paper, we used polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS), Brewster angle microscopy (BAM), and ellipsometry to investigate changes both in the structure of AnxA5 and phospholipid head groups associated with membrane binding. We found that the secondary structure of AnxA5 in the AnxA5/Ca(2+)/lipid ternary complex is conserved, mainly in alpha-helices and the average orientation of the alpha-helices of the protein is slightly tilted with respect to the normal to the phospholipid monolayer. Upon interaction between AnxA5 and phospholipids, a shift of the nu(as) PO(2)(-) band is observed by PMIRRAS. This reveals that the phosphate group is the main group involved in the binding of AnxA5 to phospholipids via Ca(2+) ions, even when some carboxylate groups are accessible (PS). PMIRRAS spectra also indicate a change of carboxylate orientation in the aspartate and glutamate residues implicated in the association of the AnxA5, which could be linked to the 2D crystallization of protein under the phospholipid monolayer. Finally, we demonstrated that the interaction of AnxA5 with pure carboxylate groups of an oleic acid monolayer is possible, but the orientation of the protein under the lipid is completely different.

  11. Crystallizing Membrane Proteins Using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Cherezov, Vadim

    2009-01-01

    A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:19390528

  12. Supported lipid bilayers as models for studying membrane domains.

    PubMed

    Kiessling, Volker; Yang, Sung-Tae; Tamm, Lukas K

    2015-01-01

    Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form "rafts" are important but overlaid with "picket-fence" interactions that are imposed by a subtended cytoskeletal network.

  13. Peroxidation of tobacco membrane lipids by the photosensitizing toxin, cercosporin.

    PubMed

    Daub, M E

    1982-06-01

    Cercosporin, a nonspecific toxin from Cercospora species, is a photosensitizing compound which rapidly kills plant cells in the light. Cell death appears to be due to a cercosporin-mediated peroxidation of membrane lipids. Tobacco leaf discs treated with cercosporin showed a large increase in electrolyte leakage 1 to 2 minutes after irradiation with light. All tobacco protoplasts exposed to cercosporin in the light were damaged within 45 minutes. Chloroform:methanol extracts of toxin-treated suspension cultures gave positive reactions for lipid hydroperoxides in the thiobarbituric acid test. Cercosporin-treated leaf discs emitted high concentrations of ethane 12 to 24 hours after incubation in the light. Cercosporin also oxidized solutions of methyl linolenate as determined by the thiobarbituric acid assay and the emission of ethane. alpha-Tocopherol had an inhibitory effect on the cercosporin-mediated lipid peroxidation.

  14. Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes.

    PubMed

    Alsop, Richard J; Toppozini, Laura; Marquardt, Drew; Kučerka, Norbert; Harroun, Thad A; Rheinstädter, Maikel C

    2015-03-01

    Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes. We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir-Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.

  15. Lipid exchange between membranes: effects of membrane surface charge, composition, and curvature.

    PubMed

    Zhu, Tao; Jiang, Zhongying; Ma, Yuqiang

    2012-09-01

    Intermembrane lipid exchange is critical to membrane functions and pharmaceutical applications. The exchange process is not fully understood and it is explored by quartz crystal microbalance with dissipation monitor method in this research. It is found that intermembrane lipid exchange is accelerated with the decrease of vesicle size and the increase of charge and liquid crystalline lipid composition ratio. Vesicle adsorption rate, membrane lateral pressure gradient, and lipid lateral diffusion coefficient are inferred to be critical in deciding the lipid exchange kinetics between membranes. Besides that, the membrane contact situation during lipid exchange is also studied. The maximum total membrane contact area is found to increase with the decrease of vesicle size, charged and liquid crystalline lipid composition ratio. A competition mechanism between the vesicle adsorption rate and the intermembrane lipid exchange rate was proposed to control the maximum total membrane contact area.

  16. Lipids: architects and regulators of membrane dynamics and trafficking.

    PubMed

    Moreau, Patrick

    2007-05-01

    We have recently shown that an inhibition of sterol synthesis by fenpropimorph leads to an accumulation of sterol precursors, hydroxypalmitic acid-containing glucosylceramides and detergent resistant membranes in the Golgi bodies instead of the plasma membrane, suggesting that the individual molecules or the microdomains were blocked in the Golgi. These results and others from several eukaryotic models link lipid metabolism with membrane morphodynamics that are involved in membrane trafficking. Focus has been expanded to other lipid families, and numerous evidences are given showing lipids and lipid-modifying enzymes as key regulators of membrane homeostasis which can strongly regulate membrane morphodynamics and therefore trafficking. Beside protein-based machineries, lipid-based machineries are also shown as crucial regulatory forces involved in protein transport and sorting.

  17. Electrophoretic separation method for membrane pore-forming proteins in multilayer lipid membranes.

    PubMed

    Okamoto, Yukihiro; Tsujimoto, Yusuke; Umakoshi, Hiroshi

    2016-03-01

    In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore-forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high-performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore-forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore-forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore-forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high-performance by utilizing the specific properties of lipid membranes.

  18. Membrane proteins, lipids and detergents: not just a soap opera.

    PubMed

    Seddon, Annela M; Curnow, Paul; Booth, Paula J

    2004-11-03

    Studying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will concentrate on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant molecular properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystallization. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.

  19. Counterion-mediated pattern formation in membranes containing anionic lipids

    PubMed Central

    Slochower, David R.; Wang, Yu-Hsiu; Tourdot, Richard W.; Radhakrishnan, Ravi; Janmey, Paul A.

    2014-01-01

    Most lipid components of cell membranes are either neutral, like cholesterol, or zwitterionic, like phosphatidylcholine and sphingomyelin. Very few lipids, such as sphingosine, are cationic at physiological pH. These generally interact only transiently with the lipid bilayer, and their synthetic analogs are often designed to destabilize the membrane for drug or DNA delivery. However, anionic lipids are common in both eukaryotic and prokaryotic cell membranes. The net charge per anionic phospholipid ranges from −1 for the most abundant anionic lipids such has phosphatidylserine, to near −7 for phosphatidylinositol 3,4,5 trisphosphate, although the effective charge depends on many environmental factors. Anionic phospholipids and other negatively charged lipids such as lipopolysaccharides are not randomly distributed in the lipid bilayer, but are highly restricted to specific leaflets of the bilayer and to regions near transmembrane proteins or other organized structures within the plane of the membrane. This review highlights some recent evidence that counterions, in the form of monovalent or divalent metal ions, polyamines, or cationic protein domains, have a large influence of the lateral distribution of anionic lipids within the membrane, and that lateral demixing of anionic lipids has effects on membrane curvature and protein function that are important for biological control. PMID:24556233

  20. Thermal Adaptation of the Archaeal and Bacterial Lipid Membranes

    PubMed Central

    Koga, Yosuke

    2012-01-01

    The physiological characteristics that distinguish archaeal and bacterial lipids, as well as those that define thermophilic lipids, are discussed from three points of view that (1) the role of the chemical stability of lipids in the heat tolerance of thermophilic organisms: (2) the relevance of the increase in the proportion of certain lipids as the growth temperature increases: (3) the lipid bilayer membrane properties that enable membranes to function at high temperatures. It is concluded that no single, chemically stable lipid by itself was responsible for the adaptation of surviving at high temperatures. Lipid membranes that function effectively require the two properties of a high permeability barrier and a liquid crystalline state. Archaeal membranes realize these two properties throughout the whole biological temperature range by means of their isoprenoid chains. Bacterial membranes meet these requirements only at or just above the phase-transition temperature, and therefore their fatty acid composition must be elaborately regulated. A recent hypothesis sketched a scenario of the evolution of lipids in which the “lipid divide” emerged concomitantly with the differentiation of archaea and bacteria. The two modes of thermal adaptation were established concurrently with the “lipid divide.” PMID:22927779

  1. Curvature forces in membrane lipid-protein interactions.

    PubMed

    Brown, Michael F

    2012-12-11

    Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes in membrane proteins, involving folding, stability, and membrane shape transitions, potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function.

  2. Curvature Forces in Membrane Lipid-Protein Interactions

    PubMed Central

    Brown, Michael F.

    2012-01-01

    Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes of membrane proteins—involving folding, stability, and membrane shape transitions—potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics, and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function. PMID:23163284

  3. Alteration of interleaflet coupling due to compounds displaying rapid translocation in lipid membranes

    PubMed Central

    Reigada, Ramon

    2016-01-01

    The spatial coincidence of lipid domains at both layers of the cell membrane is expected to play an important role in many cellular functions. Competition between the surface interleaflet tension and a line hydrophobic mismatch penalty are conjectured to determine the transversal behavior of laterally heterogeneous lipid membranes. Here, by a combination of molecular dynamics simulations, a continuum field theory and kinetic equations, I demonstrate that the presence of small, rapidly translocating molecules residing in the lipid bilayer may alter its transversal behavior by favoring the spatial coincidence of similar lipid phases. PMID:27596355

  4. The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity.

    PubMed

    Tang, Daxin; Dean, William L; Borchman, Douglas; Paterson, Christopher A

    2006-03-01

    Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.

  5. The Interaction of Polyene Antibiotics with Thin Lipid Membranes

    PubMed Central

    Andreoli, Thomas E.; Monahan, Marcia

    1968-01-01

    Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (Rm ≅ 108 ohm-cm2) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (Ti) indicate that these membranes are cation-selective (TNa ≅ 0.85; TCl ≅ 0.15). These electrical properties are independent of the cholesterol content of the lipid solutions from which the membranes are formed. Nystatin, and probably amphotericin B, are cyclic polyene antibiotics containing ≈36 ring atoms and a free amino and carboxyl group. When the lipid solutions used to form membranes contained equimolar amounts of cholesterol and phospholipid, these antibiotics reduced Rm to ≈102 ohm-cm2; concomitantly, TCl became ≅0.92. The slope of the line relating log Rm and log antibiotic concentration was ≅4.5. Neither nystatin (2 x 10-5 M) nor amphotericin B (2 x 10-7 M) had any effect on membrane stability. The antibiotics had no effect on Rm or membrane permselectivity when the lipids used to form membranes were cholesterol-depleted. Filipin (10-5 M), an uncharged polyene with 28 ring atoms, produced striking membrane instability, but did not affect Rm or membrane ionic selectivity. These data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions (Cl-, acetate), and, to a lesser degree, cations (Na+, K+, Li+). PMID:5672005

  6. Membranes: a meeting point for lipids, proteins and therapies

    PubMed Central

    Escribá, Pablo V; González-Ros, José M; Goñi, Félix M; Kinnunen, Paavo K J; Vigh, Lászlo; Sánchez-Magraner, Lissete; Fernández, Asia M; Busquets, Xavier; Horváth, Ibolya; Barceló-Coblijn, Gwendolyn

    2008-01-01

    Abstract Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy. PMID:18266954

  7. Membranes: a meeting point for lipids, proteins and therapies.

    PubMed

    Escribá, Pablo V; González-Ros, José M; Goñi, Félix M; Kinnunen, Paavo K J; Vigh, Lászlo; Sánchez-Magraner, Lissete; Fernández, Asia M; Busquets, Xavier; Horváth, Ibolya; Barceló-Coblijn, Gwendolyn

    2008-06-01

    Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.

  8. Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.

    PubMed Central

    Snel, M M; Marsh, D

    1994-01-01

    Apocytochrome c derived from horse heart cytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region of the primary sequence, and cytochrome c from yeast was spin-labeled on the single cysteine residue at sequence position 102 in the C-terminal region. The spin-labeled apocytochrome c and cytochrome c were bound to fluid bilayers composed of different negatively charged phospholipids that also contained phospholipid probes that were spin-labeled either in the headgroup or at different positions in the sn-2 acyl chain. The location of the spin-labeled cysteine residues on the lipid-bound proteins was determined relative to the spin-label positions in the different spin-labeled phospholipids by the influence of spin-spin interactions on the microwave saturation properties of the spin-label electron spin resonance spectra. The enhanced spin relaxation observed in the doubly labeled systems arises from Heisenberg spin exchange, which is determined by the accessibility of the spin-label group on the protein to that on the lipid. It is found that the labeled cysteine groups in horse heart apocytochrome c are located closest to the 14-C atom of the lipid acyl chain when the protein is bound to dimyristoyl- or dioleoyl-phosphatidylglycerol, and to that of the 5-C atom when the protein is bound to a dimyristoylphosphatidylglycerol/dimyristoylphosphatidylcholine (15:85 mol/mol mixture. On binding to dioleoylphosphatidylglycerol, the labeled cysteine residue in yeast cytochrome c is located closest to the phospholipid headgroups but possibly between the polar group region and the 5-C atom of the acyl chains. These data determine the extent to which the different regions of the proteins are able to penetrate negatively charged phospholipid bilayers. Images FIGURE 1 PMID:7948687

  9. Control of lipid membrane stability by cholesterol content.

    PubMed Central

    Raffy, S; Teissié, J

    1999-01-01

    Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content. PMID:10096902

  10. MOLECULAR GENETIC AND BIOCHEMICAL APPROACHES FOR DEFINING LIPID-DEPENDENT MEMBRANE PROTEIN FOLDING

    PubMed Central

    Dowhan, William; Bogdanov, Mikhail

    2011-01-01

    We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long–range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually depend on each other for

  11. Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

    PubMed Central

    Zech, Tobias; Ejsing, Christer S; Gaus, Katharina; de Wet, Ben; Shevchenko, Andrej; Simons, Kai; Harder, Thomas

    2009-01-01

    Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate the accumulation of specific molecular lipid species with the specific plasma membrane condensation at sites of TCR activation and with early TCR activation responses. PMID:19177148

  12. 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.

  13. 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

  14. 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.

  15. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  16. Elucidating how bamboo salt interacts with supported lipid membranes: influence of alkalinity on membrane fluidity.

    PubMed

    Jeong, Jong Hee; Choi, Jae-Hyeok; Kim, Min Chul; Park, Jae Hyeon; Herrin, Jason Scott; Kim, Seung Hyun; Lee, Haiwon; Cho, Nam-Joon

    2015-07-01

    Bamboo salt is a traditional medicine produced from sea salt. It is widely used in Oriental medicine and is an alkalizing agent with reported antiinflammatory, antimicrobial and chemotherapeutic properties. Notwithstanding, linking specific molecular mechanisms with these properties has been challenging to establish in biological systems. In part, this issue may be related to bamboo salt eliciting nonspecific effects on components found within these systems. Herein, we investigated the effects of bamboo salt solution on supported lipid bilayers as a model system to characterize the interaction between lipid membranes and bamboo salt. The atomic composition of unprocessed and processed bamboo salts was first analyzed by mass spectrometry, and we identified several elements that have not been previously reported in other bamboo salt preparations. The alkalinity of hydrated samples was also measured and determined to be between pH 10 and 11 for bamboo salts. The effect of processed bamboo salt solutions on the fluidic properties of a supported lipid bilayer on glass was next investigated by fluorescence recovery after photobleaching (FRAP) analysis. It was demonstrated that, with increasing ionic strength of the bamboo salt solution, the fluidity of a lipid bilayer increased. On the contrary, increasing the ionic strength of near-neutral buffer solutions with sodium chloride salt diminished fluidity. To reconcile these two observations, we identified that solution alkalinity is critical for the effects of bamboo salt on membrane fluidity, as confirmed using three additional commercial bamboo salt preparations. Extended-DLVO model calculations support that the effects of bamboo salt on lipid membranes are due to the alkalinity imparting a stronger hydration force. Collectively, the results of this work demonstrate that processing of bamboo salt strongly affects its atomic composition and that the alkalinity of bamboo salt solutions contributes to its effect on membrane

  17. The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis.

    PubMed

    Somerharju, Pentti; Virtanen, Jorma A; Cheng, Kwan H; Hermansson, Martin

    2009-01-01

    Most biological membranes are extremely complex structures consisting of hundreds of different lipid and protein molecules. According to the famous fluid-mosaic model lipids and many proteins are free to diffuse very rapidly in the plane of the membrane. While such fast diffusion implies that different membrane lipids would be laterally randomly distributed, accumulating evidence indicates that in model and natural membranes the lipid components tend to adopt regular (superlattice-like) distributions. The superlattice model, put forward based on such evidence, is intriguing because it predicts that 1) there is a limited number of allowed compositions representing local minima in membrane free energy and 2) those energy minima could provide set-points for enzymes regulating membrane lipid compositions. Furthermore, the existence of a discrete number of allowed compositions could help to maintain organelle identity in the face of rapid inter-organelle membrane traffic.

  18. Membrane lipids and sodium pumps of cattle and crocodiles: an experimental test of the membrane pacemaker theory of metabolism.

    PubMed

    Wu, B J; Hulbert, A J; Storlien, L H; Else, P L

    2004-09-01

    The influence of membrane lipid composition on the molecular activity of a major membrane protein (the sodium pump) was examined as a test of the membrane pacemaker theory of metabolism. Microsomal membranes from the kidneys of cattle (Bos taurus) and crocodiles (Crocodylus porosus) were found to possess similar sodium pump concentrations, but cattle membranes showed a four- to fivefold higher enzyme (Na(+)-K(+)-ATPase) activity when measured at 37 degrees C. The molecular activity of the sodium pumps (ATP/min) from both species was fully recoverable when delipidated pumps were reconstituted with membrane from the original source (same species). The results of experiments involving species membrane crossovers showed cattle sodium pump molecular activity to progressively decrease from 3,245 to 1,953 (P < 0.005) to 1,031 (P < 0.003) ATP/min when subjected to two cycles of delipidation and reconstitution with crocodile membrane as a lipid source. In contrast, the molecular activity of crocodile sodium pumps progressively increased from 729 to 908 (P < 0.01) to 1,476 (P = 0.01) ATP/min when subjected to two cycles of delipidation and reconstitution with cattle membrane as a lipid source. The lipid composition of the two membrane preparations showed similar levels of saturated ( approximately 31-34%) and monounsaturated ( approximately 23-25%) fatty acids. Cattle membrane had fourfold more n-3 polyunsaturated fatty acids (11.2 vs. 2.9%) but had a reduced n-6 polyunsaturate content (29 vs. 43%). The results support the membrane pacemaker theory of metabolism and suggest membrane lipids and their polyunsaturates play a significant role in determining the molecular activity of the sodium pump.

  19. Effects of dimethyl sulfoxide on lipid membrane electroporation.

    PubMed

    Fernández, M Laura; Reigada, Ramon

    2014-08-07

    Pores can be generated in lipid membranes by the application of an external electric field or by the addition of particular chemicals such as dimethyl sulfoxide (DMSO). Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes in both situations. By means of MD simulations, we address the formation of electropores in cholesterol-containing lipid bilayers under the influence of DMSO. We show how a combination of physical and chemical mechanisms leads to more favorable conditions for generating membrane pores and, in particular, how the addition of DMSO to the medium significantly reduces the minimum electric field required to electroporate a lipid membrane. The strong alteration of membrane transversal properties and the energetic stabilization of the hydrophobic pore stage by DMSO provide the physicochemical mechanisms that explain this effect.

  20. Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids.

    PubMed

    Raghavan, Vijay; Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Yamada, Masayoshi; Morisada, Megan; Labhasetwar, Vinod

    2015-10-27

    Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport

  1. New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

    PubMed Central

    Mateo, C R; Souto, A A; Amat-Guerri, F; Acuña, A U

    1996-01-01

    The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A. Images FIGURE 10 PMID:8889194

  2. Nucleic acid-lipid membrane interactions studied by DSC.

    PubMed

    Giatrellis, Sarantis; Nounesis, George

    2011-01-01

    The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA-lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid-membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies.

  3. Membrane proteins bind lipids selectively to modulate their structure and function

    PubMed Central

    Allison, Timothy M.; Ulmschneider, Martin B.; Degiacomi, Matteo T.; Baldwin, Andrew J.; Robinson, Carol V.

    2014-01-01

    Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1-7 and that lipids can bind to specific sites, for example in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IM-MS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these

  4. Lipids in the assembly of membrane proteins and organization of protein supercomplexes: implications for lipid-linked disorders.

    PubMed

    Bogdanov, Mikhail; Mileykovskaya, Eugenia; Dowhan, William

    2008-01-01

    Lipids play important roles in cellular dysfunction leading to disease. Although a major role for phospholipids is in defining the membrane permeability barrier, phospholipids play a central role in a diverse range of cellular processes and therefore are important factors in cellular dysfunction and disease. This review is focused on the role of phospholipids in normal assembly and organization of the membrane proteins, multimeric protein complexes, and higher order supercomplexes. Since lipids have no catalytic activity, it is difficult to determine their function at the molecular level. Lipid function has generally been defined by affects on protein function or cellular processes. Molecular details derived from genetic, biochemical, and structural approaches are presented for involvement of phosphatidylethanolamine and cardiolipin in protein organization. Experimental evidence is presented that changes in phosphatidylethanolamine levels results in misfolding and topological misorientation of membrane proteins leading to dysfunctional proteins. Examples are presented for diseases in which proper protein folding or topological organization is not attained due to either demonstrated or proposed involvement of a lipid. Similar changes in cardiolipin levels affects the structure and function of individual components of the mitochondrial electron transport chain and their organization into supercomplexes resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to reduced cardiolipin levels are described. Therefore, understanding the principles governing lipid-dependent assembly and organization of membrane proteins and protein complexes will be useful in developing novel therapeutic approaches for disorders in which lipids play an important role.

  5. Interaction of LL-37 with Model Membrane Systems of Different Complexity: Influence of the Lipid Matrix

    PubMed Central

    Sevcsik, E.; Pabst, G.; Richter, W.; Danner, S.; Amenitsch, H.; Lohner, K.

    2008-01-01

    As the main difference between bacterial and mammalian cell membranes is their net charge, the focal point of consideration in many model membrane experiments with antimicrobial peptides is lipid headgroup charge. We studied the interaction of the human multifunctional peptide LL-37 with single phospholipid monolayers, bilayers, and bilayers composed of binary mixtures of the four phospholipid species predominantly used in model membrane experiments (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine). We found that 1), the effects on single lipid monolayers are not comparable to those on the corresponding bilayers; 2), there are four different effects of LL-37 on bilayers of the four lipids; 3), the preference of LL-37 for the specific lipids is roughly inversely related to chain packing density; and 4), in the binary lipid mixtures, one lipid—and not necessarily the charged one—generally governs the mode of lipid/peptide interaction. Thus, our results show that lipid net charge is not the decisive factor determining the membrane-perturbing mechanism of LL-37, but only one of several parameters, among them packing density, the ability to form intermolecular H-bonds, and lipid molecular shape, which emphasizes how profoundly the choice of the model system can influence the outcome of a study of lipid/peptide interaction. PMID:18326643

  6. Differential Effect of Plant Lipids on Membrane Organization

    PubMed Central

    Grosjean, Kevin; Mongrand, Sébastien; Beney, Laurent; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2015-01-01

    The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains. PMID:25575593

  7. The electrical interplay between proteins and lipids in membranes.

    PubMed

    Richens, Joanna L; Lane, Jordan S; Bramble, Jonathan P; O'Shea, Paul

    2015-09-01

    All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure. The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid-protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid-protein interactions.

  8. Electro-hydrodynamic effects on lipid membranes in giant vesicles

    NASA Astrophysics Data System (ADS)

    Staykova, Margarita; Yamamoto, Tetsuya; Lipowsky, Reinhard; Dimova, Rumiana

    2009-11-01

    Electric fields are widely applied for cell manipulation in numerous micron-scale systems. Here, we show for the first time that alternating electric fields may cause pronounced flows in the membrane of giant lipid vesicles as well as in the surrounding fluid media.^ The lipid vesicles are not only biomimetic model for the cell membrane but also have many potential biotechnological applications, e.g. as drug-delivery systems and micro-reactors. The reported effects should be considered in electric micro-manipulation procedures on cells and vesicles. They might be useful for applications in microfluidic technologies, for lipid mixing, trapping and displacement, as will be demonstrated. We also believe that our method for visualization of the lipid flows by fluorescently labeled intra-membrane domains will be helpful for studies on membrane behavior in vesicles subjected to shear or mechanical stresses.

  9. Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane.

    PubMed

    Tiriveedhi, Venkataswarup; Miller, Melissa; Butko, Peter; Li, Min

    2012-07-01

    The S4 transmembrane segment in voltage-gated ion channels, a highly basic alpha helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 +/- 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 A. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the at helicity and regular spacing of basic amino-acid residues in the S4 sequence.

  10. Autonomous Transmembrane Segment S4 of the Voltage Sensor Domain Partitions into the Lipid Membrane

    PubMed Central

    Tiriveedhi, Venkataswarup; Miller, Melissa; Butko, Peter; Li, Min

    2012-01-01

    The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 ± 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 Å. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence. PMID:22465069

  11. Lipid partitioning at the nuclear envelope controls membrane biogenesis

    PubMed Central

    Barbosa, Antonio Daniel; Sembongi, Hiroshi; Su, Wen-Min; Abreu, Susana; Reggiori, Fulvio; Carman, George M.; Siniossoglou, Symeon

    2015-01-01

    Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage. PMID:26269581

  12. Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition

    PubMed Central

    Banquy, Xavier; Kristiansen, Kai; Lee, Dong Woog; Israelachvili, Jacob N.

    2012-01-01

    We report the effects of calcium ions on the adhesion and hemifusion mechanisms of model supported myelin lipid bilayer membranes of differing lipid composition. As in our previous studies [1, 2], the lipid compositions used mimic “healthy” and “diseased-like” (experimental autoimmune encephalomyelitis, EAE) membranes. Our results show that the interaction forces as a function of membrane separation distance are well described by a generic model that also (and in particular) includes the hydrophobic interaction arising from the hydrophobically exposed (interior) parts of the bilayers. The model is able to capture the mechanical instability that triggers the onset of the hemifusion event, and highlights the primary role of the hydrophobic interaction in membrane fusion. The effects of lipid composition on the fusion mechanism, and the adhesion forces between myelin lipid bilayers, can be summarized as follow: in calcium-free buffer, healthy membranes do not present any signs of adhesion or hemifusion, while diseased membranes hemifuse easily. Addition of 2 mM calcium favors adhesion and hemifusion of the membranes independently of their composition, but the mechanisms involved in the two processes were different: healthy bilayers systematically presented stronger adhesion forces and lower energy barriers to fusion compared to diseased bilayers. These results are of particular relevance for understanding lesion development (demyelination, swelling, vacuolization and/or vesiculation) in myelin associated diseases such as multiple sclerosis and its relationship to lipid domain formation in myelin membranes. PMID:22047743

  13. Lipid rafts and detergent-resistant membranes in epithelial keratinocytes.

    PubMed

    McGuinn, Kathleen P; Mahoney, Mỹ G

    2014-01-01

    Our understanding of the plasma membrane has markedly increased since Singer and Nicolson proposed the fluid mosaic model in 1972. While their revolutionary theory of the lipid bilayer remains largely valid, it is now known that lipids and proteins are not randomly dispersed throughout the plasma membrane but instead may be organized within membrane microdomains, commonly referred to as lipid rafts. Lipid rafts are highly dynamic, detergent resistant, and enriched with both cholesterol and glycosphingolipids. The two main types are flotillin-rich planar lipid rafts and caveolin-rich caveolae. It is proposed that flotillin and caveolin proteins regulate cell communication by compartmentalizing and interacting with signal transduction proteins within their respective lipid microdomains. Consequently, membrane rafts play an important role in vital cellular functions including migration, invasion, and signaling; thus, alterations in their microenvironment can initiate signaling pathways that affect cellular function and behavior. Therefore, the identification of lipid rafts and their associated proteins is integral to the study of transmembrane signaling. Here, we review the current standard protocols and biochemical approaches used to isolate and define raft proteins from epithelial cells and tissues. Furthermore, in Section 3 of this chapter, detailed protocols are offered for isolating lipid rafts by subjection to detergent and sucrose density centrifugation, as well as an approach for selectively isolating caveolae. Methods to manipulate rafts with treatments such as methyl-β-cyclodextrin and flotillin III are also described.

  14. Lipid flow through fusion pores connecting membranes of different tensions.

    PubMed

    Chizmadzhev, Y A; Kumenko, D A; Kuzmin, P I; Chernomordik, L V; Zimmerberg, J; Cohen, F S

    1999-06-01

    When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores.

  15. Lipid flow through fusion pores connecting membranes of different tensions.

    PubMed Central

    Chizmadzhev, Y A; Kumenko, D A; Kuzmin, P I; Chernomordik, L V; Zimmerberg, J; Cohen, F S

    1999-01-01

    When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores. PMID:10354423

  16. Ca(2+) adsorption to lipid membranes and the effect of cholesterol in their composition.

    PubMed

    Iraolagoitia, Ximena L Raffo; Martini, M Florencia

    2010-03-01

    The aim of this work is to determine the binding of ionic calcium (Ca(2+)) to lipid membranes in which the availability of the phosphate groups to the aqueous phase is modified by the degree of saturation of the lipids and the inclusion of cholesterol. The shifts in the phosphate bands observed in the Fourier transform infrared spectroscopy (FTIR) spectra are direct evidence of the interaction of Ca(2+) with phosphate groups. The binding analysis was done by determining the changes in the zeta potential of liposomes suspended in buffer at controlled temperature. The changes produced by the ion on the zeta potential of dioleoylphosphatidylcholine (DOPC); dipalmitoylphosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC); dimyristoylphosphatidylethanolamine (DMPE) and their mixers with cholesterol were measured, showing a Langmuir isotherm behavior in all the lipid composition assayed. The results show that the interaction of Ca(2+) to lipid membranes depends on the exposure and the density of phosphate groups at the membrane interphase.

  17. Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature

    PubMed Central

    Cheney, Philip P.; Weisgerber, Alan W.; Feuerbach, Alec M.; Knowles, Michelle K.

    2017-01-01

    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. PMID:28294967

  18. Pushing the lipid envelope: using bio-inspired nanocomposites to understand and exploit lipid membrane limitations

    NASA Astrophysics Data System (ADS)

    Montano, Gabriel

    Lipids serve as the organizing matrix material for biological membranes, the site of interaction of cells with the external environment. . As such, lipids play a critical role in structure/function relationships of an extraordinary number of critical biological processes. In this talk, we will look at bio-inspired membrane assemblies to better understand the roles of lipids in biological systems as well as attempt to generate materials that can mimic and potentially advance upon biological membrane processes. First, we will investigate the response of lipids to adverse conditions. In particular, I will present data that demonstrates the response of lipids to harsh conditions and how such responses can be exploited to generate nanocomposite rearrangements. I will also show the effect of adding the endotoxin lipopolysaccharide (LPS) to lipid bilayer assemblies and describe implications on our understanding of LPS organization in biological systems as well as describe induced lipid modifications that can be exploited to organize membrane composites with precise, two-dimensional geometric control. Lastly, I will describe the use of amphiphilic block copolymers to create membrane nanocomposites capable of mimicking biological systems. In particular, I will describe the use of our polymer-based membranes in creating artificial photosynthetic assemblies that rival biological systems in function in a more flexible, dynamic matrix.

  19. Probing the Lipid Membrane Dipole Potential by Atomic Force Microscopy

    PubMed Central

    Yang, Yi; Mayer, Kathryn M.; Wickremasinghe, Nissanka S.; Hafner, Jason H.

    2008-01-01

    The electrostatic properties of biological membranes can be described by three parameters: the transmembrane potential, the membrane surface potential, and the membrane dipole potential. The first two are well characterized in terms of their magnitudes and biological effects. The dipole potential, however, is not well characterized. Various methods to measure the membrane dipole potential indirectly yield different values, and there is not even agreement on the source of the membrane dipole moment. This ambiguity impedes investigations into the biological effects of the membrane dipole moment, which should be substantial considering the large interfacial fields with which it is associated. Electrostatic analysis of phosphatidylcholine lipid membranes with the atomic force microscope reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipids. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internal membrane dipole potential. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported phosphatidylcholine membranes. This new ability to quantitatively measure the membrane dipole moment in a noninvasive manner with nanometer scale spatial resolution will be useful in identifying the biological effects of the dipole potential. PMID:18805919

  20. Mechanisms of membrane deformation by lipid-binding domains.

    PubMed

    Itoh, Toshiki; Takenawa, Tadaomi

    2009-09-01

    Among an increasing number of lipid-binding domains, a group that not only binds to membrane lipids but also changes the shape of the membrane has been found. These domains are characterized by their strong ability to transform globular liposomes as well as flat plasma membranes into elongated membrane tubules both in vitro and in vivo. Biochemical studies on the structures of these proteins have revealed the importance of the amphipathic helix, which potentially intercalates into the lipid bilayer to induce and/or sense membrane curvature. Among such membrane-deforming domains, BAR and F-BAR/EFC domains form crescent-shaped dimers, suggesting a preference for a curved membrane, which is important for curvature sensing. Bioinformatics in combination with structural analyses has been identifying an increasing number of novel families of lipid-binding domains. This review attempts to summarize the evidence obtained by recent studies in order to gain general insights into the roles of membrane-deforming domains in a variety of biological events.

  1. 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

  2. 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.

  3. Characterization of Membrane Protein-Lipid Interactions by Mass Spectrometry Ion Mobility Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Cong, Xiao; Liu, Wen; Laganowsky, Arthur

    2016-12-01

    Lipids in the biological membrane can modulate the structure and function of integral and peripheral membrane proteins. Distinguishing individual lipids that bind selectively to membrane protein complexes from an ensemble of lipid-bound species remains a daunting task. Recently, ion mobility mass spectrometry (IM-MS) has proven to be invaluable for interrogating the interactions between protein and individual lipids, where the complex undergoes collision induced unfolding followed by quantification of the unfolding pathway to assess the effect of these interactions. However, gas-phase unfolding experiments for membrane proteins are typically performed on the entire ensemble (apo and lipid bound species), raising uncertainty to the contribution of individual lipids and the species that are ejected in the unfolding process. Here, we describe the application of mass spectrometry ion mobility mass spectrometry (MS-IM-MS) for isolating ions corresponding to lipid-bound states of a model integral membrane protein, ammonia channel (AmtB) from Escherichia coli. Free of ensemble effects, MS-IM-MS reveals that bound lipids are ejected as neutral species; however, no correlation was found between the lipid-induced stabilization of complex and their equilibrium binding constants. In comparison to data obtained by IM-MS, there are surprisingly limited differences in stability measurements from IM-MS and MS-IM-MS. The approach described here to isolate ions of membrane protein complexes will be useful for other MS methods, such as surface induced dissociation or collision induced dissociation to determine the stoichiometry of hetero-oligomeric membrane protein complexes.

  4. The effect of copper ions on the lipid composition of subcellular membranes in Hydrilla verticillata.

    PubMed

    Rozentsvet, Olga A; Nesterov, Viktor N; Sinyutina, Natalia F

    2012-09-01

    The paper studies changes in the content and composition of lipids in the membranes of chloroplasts, mitochondria and microsomes of the aquatic plant Hydrilla verticillata exposed to copper ions (100 μM; 1, 3, 6 and 24 h). The rate of copper accumulation and the coefficient of its extraction by the plant were also determined. The presence of copper in the incubation medium and its accumulation in the plant tissues decreased the content of photosynthetic pigments, stimulated lipid peroxidation and enhanced membrane permeability. The gradual accumulation of copper in the plant tissues was accompanied by specific changes in the composition of lipids: the content of sulfolipids (SQDG) in chloroplasts declined; the content of monogalactosyl diacylglycerols (MGDG), digalactosyl diacylglycerols (DGDG) and phosphatidyl glycerols (PG) in chloroplasts and mitochondria grew after an hour of copper exposure; and the content of all the lipids except phosphatidic acids (PA) decreased after 3 h of exposure. The decline in the content of phosphatidyl cholines (PC) was first observed in the membranes of microsomes (after an hour of exposure) and later in the membranes of chloroplasts and mitochondria (after 3-6 h of exposure). The experiments with incorporation of [2-(14)C]sodium acetate into fatty acids of polar lipids showed that in parallel with lipid destruction, there took place an intensive and specific renewal of the lipid pool of subcellular membrane fractions.

  5. Red cell membrane lipid changes at 3,500 m and on return to sea level.

    PubMed

    González, Gustavo; Celedón, Gloria; Escobar, Marcela; Sotomayor, Carlos; Ferrer, Verónica; Benítez, Dixan; Behn, Claus

    2005-01-01

    Previous studies have shown that acute hypobaric hypoxia, obtained in a hypobaric chamber, and subsequent reoxygenation, give rise to modifications of the erythrocyte membrane lipid dynamics, resulting in an increased lateral diffusivity of the membrane lipids, and this was interpreted as the result of a modified lipid-protein interaction. The aim of the present study was to determine the effect of the reoxygenation condition in individuals after 3 days at an altitude of 3,500 m above sea level. Reoxygenation was a consequence of returning to sea level. Resting blood samples from both conditions were obtained, and erythrocytes were separated and immediately lysed for membrane isolation. We measured the bilayer polarity in membranes with Laurdan, a fluorescent probe. We also measured malondialdehyde in membrane lipids, an indicator of oxidative damage. We found a 12% (p = 0.016, n = 7) increase in the polarity of the membrane bilayer surface, and an increase of 70% (p = 0.005, n = 7) in the formation of malondialdehyde in the membrane after the reoxygenation condition. The membrane bilayer polarity increase is due to an oxidative modification of the phospholipid backbone after reoxygenation. People working and/or recreating at moderate altitude (3,500 m) may be at risk of erythrocyte membrane oxidative damage upon returning to sea level, and therefore a better understanding of the processes occurring upon reoxygenation may lead to proposed strategies to minimize this effect.

  6. Plasma Membrane Lipids and Their Role in Fungal Virulence

    PubMed Central

    Del Poeta, Maurizio

    2016-01-01

    There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains has been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies. PMID:26703191

  7. Interactions of Lipid Membranes with Fibrillar Protein Aggregates.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya; Girych, Mykhailo; Adachi, Emi; Mizuguchi, Chiharu; Saito, Hiroyuki

    2015-01-01

    Amyloid fibrils are an intriguing class of protein aggregates with distinct physicochemical, structural and morphological properties. They display peculiar membrane-binding behavior, thus adding complexity to the problem of protein-lipid interactions. The consensus that emerged during the past decade is that amyloid cytotoxicity arises from a continuum of cross-β-sheet assemblies including mature fibrils. Based on literature survey and our own data, in this chapter we address several aspects of fibril-lipid interactions, including (i) the effects of amyloid assemblies on molecular organization of lipid bilayer; (ii) competition between fibrillar and monomeric membrane-associating proteins for binding to the lipid surface; and (iii) the effects of lipids on the structural morphology of fibrillar aggregates. To illustrate some of the processes occurring in fibril-lipid systems, we present and analyze fluorescence data reporting on lipid bilayer interactions with fibrillar lysozyme and with the N-terminal 83-residue fragment of amyloidogenic mutant apolipoprotein A-I, 1-83/G26R/W@8. The results help understand possible mechanisms of interaction and mutual remodeling of amyloid fibers and lipid membranes, which may contribute to amyloid cytotoxicity.

  8. The adrenal specific toxicant mitotane directly interacts with lipid membranes and alters membrane properties depending on lipid composition.

    PubMed

    Scheidt, Holger A; Haralampiev, Ivan; Theisgen, Stephan; Schirbel, Andreas; Sbiera, Silviu; Huster, Daniel; Kroiss, Matthias; Müller, Peter

    2016-06-15

    Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects.

  9. Stabilization of concentration fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Palmieri, Benoit; Safran, Samuel

    2012-02-01

    Finite-size domains have been observed at the surface of cells. These lipids ``rafts'' are stable nanodomains enriched in saturated lipids and cholesterol. While line tension favors macrodomains, one explanation for raft stabilization suggests that the membrane composition is tuned close to a spinodal temperature. From this point of view, rafts are long-lived concentration fluctuations in the mixed phase. We propose a ternary mixture model for the cell membrane that includes hybrid lipids which have one saturated and one unsaturated hydrocarbon chain. Finite amount of hybrid lipids reduces the packing incompatibility at the saturated/unsaturated lipid interface and stabilizes the concentration fluctuations. Hybrid-Hybrid interactions are included in the model and further increase the life-time of the rafts and decrease their length-scales. Moreover, the hybrid has extra orientational degrees of freedom that may lead to modulated phases.

  10. Why Hydrophilic Water can Permeate Hydrophobic Interior of Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Qiao, Baofu; Olvera de La Cruz, Monica

    2014-03-01

    Water molecules as well as some small molecules have long been found to be able to diffuse across lipid membranes. Such permeation is of significant biological and biotechnological importance. For instance, the permeation of water across lipid membrane plays a important role in regulating ionic concentrations inside of cells. Such water permeation without the assistance of proteins embedded in membranes has been found to be a energetically unfavorable process. We, for the first time, explicitly depict the driving force for such an energetically unfavorable process. Atomistic molecular dynamics simulations are employed to investigate water diffusion in both liquid-crystalline and ordered gel phases of membranes containing zwitterionic DPPC or anionic DLPS lipid. The membrane conformation is calculated to have a critical role in water permeation, regardless of the type of lipid. The fluctuations in the potential energy are found to have a significant, if not the exclusive, role in the transportation of water across lipid membranes. Our results are also informative for the diffusion of small molecules of CO2, O2 and drug molecules, the absence of diffusion of ions, and the diffusion of water into the hydrophobic pores of carbon nanotubes. The authors acknowledge the support from the Office of the Director of Defense Research and Engineering (DDR & E) under Award No. FA9550-10-1-0167.

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

    PubMed

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

    2014-12-01

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

  12. Edelfosine and miltefosine effects on lipid raft properties: membrane biophysics in cell death by antitumor lipids.

    PubMed

    Castro, Bruno M; Fedorov, Aleksander; Hornillos, Valentin; Delgado, Javier; Acuña, A Ulises; Mollinedo, Faustino; Prieto, Manuel

    2013-07-03

    Edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-phosphocholine) and miltefosine (hexadecylphosphocholine) are synthetic alkylphospholipids (ALPs) that are reported to selectively accumulate in tumor cell membranes, inducing Fas clustering and activation on lipid rafts, triggering apoptosis. However, the exact mechanism by which these lipids elicit these events is still not fully understood. Recent studies propose that their mode of action might be related with alterations of lipid rafts biophysical properties caused by these lipid drugs. To achieve a clear understanding of this mechanism, we studied the effects of pharmacologically relevant amounts of edelfosine and miltefosine in the properties of model and cellular membranes. The influence of these molecules on membrane order, lateral organization, and lipid rafts molar fraction and size were studied by steady-state and time-resolved fluorescence methods, Förster resonance energy transfer (FRET), confocal and fluorescence lifetime imaging microscopy (FLIM). We found that the global membrane and lipid rafts biophysical properties of both model and cellular membranes were not significantly affected by both the ALPs. Nonetheless, in model membranes, a mild increase in membrane fluidity induced by both alkyl lipids was detected, although this effect was more noticeable for edelfosine than miltefosine. This absence of drastic alterations shows for the first time that ALPs mode of action is unlikely to be directly linked to alterations of lipid rafts biophysical properties caused by these drugs. The biological implications of this result are discussed in the context of ALPs effects on lipid metabolism, mitochondria homeostasis modulation, and their relationship with tumor cell death.

  13. Lipid and fatty acid composition of Gluconobacter oxydans before and after intracytoplasmic membrane formation.

    PubMed Central

    Heefner, D L; Claus, G W

    1978-01-01

    Gluconobacter oxydans differentiates by forming quantities of intracytoplasmic membranes at the end of exponential growth, and this formation occurs concurrently with a 60% increase in cellular lipid. The present study was initiated to determine whether this newly synthesized lipid differed from that extracted before intracytoplasmic membrane synthesis. Undifferentiated exponential-phase cells were found to contain 30% phosphatidylcholine, 27.1% caridolipin, 25% phosphatidylethanolamine, 12.5% phosphatidylglycerol, 0.4% phosphatidic acid, 0.2% phosphatidylserine, and four additional unidentified lipids totaling less than 5%. The only change detected after formation of intracytoplasmic membranes was a slight decrease in phosphatidylethanolamine and a corresponding increase in phosphatidylcholine. An examination of lipid hydrolysates revealed 11 different fatty acids in the lipids from each cell type. Hexadecanoic acid and monounsaturated octadecenoic accounted for more than 75% of the total fatty acids for both cell types. Proportional changes were noted in all fatty acids except octadecenoate. Anteiso-pentadecanoate comprised less than 1% of the fatty acids from undifferentiated cells but more than 13% of the total fatty acids from cells containing intracytoplasmic membranes. These results suggest that anteiso-pentadecanoate formation closely parallels the formation of intracytoplasmic membranes. Increased concentrations of this fatty acid may contribute to the fluidity necessary for plasma membrane convolution during intracytoplasmic membrane development. PMID:649571

  14. Lipid domains in model membranes: a brief historical perspective.

    PubMed

    Mouritsen, Ole G; Bagatolli, Luis A

    2015-01-01

    All biological membranes consist of a complex composite of macromolecules and macromolecular assemblies, of which the fluid lipid-bilayer component is a core element with regard to cell encapsulation and barrier properties. The fluid lipid bilayer also supports the functional machinery of receptors, channels and pumps that are associated with the membrane. This bilayer is stabilized by weak physical and colloidal forces, and its nature is that of a self-assembled system of amphiphiles in water. Being only approximately 5 nm in thickness and still encapsulating a cell that is three orders of magnitude larger in diameter, the lipid bilayer as a material has very unusual physical properties, both in terms of structure and dynamics. Although the lipid bilayer is a fluid, it has a distinct and structured trans-bilayer profile, and in the plane of the bilayer the various molecular components, viz different lipid species and membrane proteins, have the capacity to organize laterally in terms of differentiated domains on different length and time scales. These elements of small-scale structure and order are crucial for the functioning of the membrane. It has turned out to be difficult to quantitatively study the small-scale structure of biological membranes. A major part of the insight into membrane micro- and nano-domains and the concepts used to describe them have hence come from studies of simple lipid bilayers as models of membranes, by use of a wide range of theoretical, experimental and simulational approaches. Many questions remain to be answered as to which extent the result from model studies can carry over to real biological membranes.

  15. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    -bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. PMID:26912608

  16. Helix-helix interactions in membrane domains of bitopic proteins: Specificity and role of lipid environment.

    PubMed

    Bocharov, Eduard V; Mineev, Konstantin S; Pavlov, Konstantin V; Akimov, Sergey A; Kuznetsov, Andrey S; Efremov, Roman G; Arseniev, Alexander S

    2017-04-01

    Interaction between transmembrane helices often determines biological activity of membrane proteins. Bitopic proteins, a broad subclass of membrane proteins, form dimers containing two membrane-spanning helices. Some aspects of their structure-function relationship cannot be fully understood without considering the protein-lipid interaction, which can determine the protein conformational ensemble. Experimental and computer modeling data concerning transmembrane parts of bitopic proteins are reviewed in the present paper. They highlight the importance of lipid-protein interactions and resolve certain paradoxes in the behavior of such proteins. Besides, some properties of membrane organization provided a clue to understanding of allosteric interactions between distant parts of proteins. Interactions of these kinds appear to underlie a signaling mechanism, which could be widely employed in the functioning of many membrane proteins. Treatment of membrane proteins as parts of integrated fine-tuned proteolipid system promises new insights into biological function mechanisms and approaches to drug design. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

  17. Applications of Mass Spectrometry to Lipids and Membranes

    PubMed Central

    Harkewicz, Richard; Dennis, Edward A.

    2012-01-01

    Lipidomics, a major part of metabolomics, constitutes the detailed analysis and global characterization, both spatial and temporal, of the structure and function of lipids (the lipidome) within a living system. As with proteomics, mass spectrometry has earned a central analytical role in lipidomics, and this role will continue to grow with technological developments. Currently, there exist two mass spectrometry-based lipidomics approaches, one based on a division of lipids into categories and classes prior to analysis, the “comprehensive lipidomics analysis by separation simplification” (CLASS), and the other in which all lipid species are analyzed together without prior separation, shotgun. In exploring the lipidome of various living systems, novel lipids are being discovered, and mass spectrometry is helping characterize their chemical structure. Deuterium exchange mass spectrometry (DXMS) is being used to investigate the association of lipids and membranes with proteins and enzymes, and imaging mass spectrometry (IMS) is being applied to the in situ analysis of lipids in tissues. PMID:21469951

  18. Ceramides in the skin lipid membranes: length matters.

    PubMed

    Skolová, Barbora; Janůšová, Barbora; Zbytovská, Jarmila; Gooris, Gert; Bouwstra, Joke; Slepička, Petr; Berka, Pavel; Roh, Jaroslav; Palát, Karel; Hrabálek, Alexandr; Vávrová, Kateřina

    2013-12-17

    Ceramides are essential constituents of the skin barrier that allow humans to live on dry land. Reduced levels of ceramides have been associated with skin diseases, e.g., atopic dermatitis. However, the structural requirements and mechanisms of action of ceramides are not fully understood. Here, we report the effects of ceramide acyl chain length on the permeabilities and biophysics of lipid membranes composed of ceramides (or free sphingosine), fatty acids, cholesterol, and cholesterol sulfate. Short-chain ceramides increased the permeability of the lipid membranes compared to a long-chain ceramide with maxima at 4-6 carbons in the acyl. By a combination of differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray diffraction, Langmuir monolayers, and atomic force microscopy, we found that the reason for this effect in short ceramides was a lower proportion of tight orthorhombic packing and phase separation of continuous short ceramide-enriched domains with shorter lamellar periodicity compared to native long ceramides. Thus, long acyl chains in ceramides are essential for the formation of tightly packed impermeable lipid lamellae. Moreover, the model skin lipid membranes are a valuable tool to study the relationships between the lipid structure and composition, lipid organization, and the membrane permeability.

  19. Structure formation of lipid membranes: Membrane self-assembly and vesicle opening-up to octopus-like micelles

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2013-02-01

    We briefly review our recent studies on self-assembly and vesicle rupture of lipid membranes using coarse-grained molecular simulations. For single component membranes, lipid molecules self-assemble from random gas states to vesicles via disk-shaped clusters. Clusters aggregate into larger clusters, and subsequently the large disks close into vesicles. The size of vesicles are determined by kinetics than by thermodynamics. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle called bicelle can be formed. When both surfactants have negligibly low critical micelle concentration, it is found that bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and spontaneous curvature of the membrane monolayer.

  20. 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.

  1. Digital holographic microscopy of phase separation in multicomponent lipid membranes

    NASA Astrophysics Data System (ADS)

    Farzam Rad, Vahideh; Moradi, Ali-Reza; Darudi, Ahmad; Tayebi, Lobat

    2016-12-01

    Lateral in-homogeneities in lipid compositions cause microdomains formation and change in the physical properties of biological membranes. With the presence of cholesterol and mixed species of lipids, phospholipid membranes segregate into lateral domains of liquid-ordered and liquid-disordered phases. Coupling of two-dimensional intralayer phase separations and interlayer liquid-crystalline ordering in multicomponent membranes has been previously demonstrated. By the use of digital holographic microscopy (DHMicroscopy), we quantitatively analyzed the volumetric dynamical behavior of such membranes. The specimens are lipid mixtures composed of sphingomyelin, cholesterol, and unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine. DHMicroscopy in a transmission mode is an effective tool for quantitative visualization of phase objects. By deriving the associated phase changes, three-dimensional information on the morphology variation of lipid stacks at arbitrary time scales is obtained. Moreover, the thickness distribution of the object at demanded axial planes can be obtained by numerical focusing. Our results show that the volume evolution of lipid domains follows approximately the same universal growth law of previously reported area evolution. However, the thickness of the domains does not alter significantly by time; therefore, the volume evolution is mostly attributed to the changes in area dynamics. These results might be useful in the field of membrane-based functional materials.

  2. Laurdan fluorescence senses mechanical strain in the lipid bilayer membrane.

    PubMed

    Zhang, Yan-Liang; Frangos, John A; Chachisvilis, Mirianas

    2006-09-01

    The precise molecular mechanisms by which cells transduce a mechanical stimulus into an intracellular biochemical response have not yet been established. Here, we show for the first time that the fluorescence emission of an environment-sensitive membrane probe Laurdan is modulated by mechanical strain of the lipid bilayer membrane. We have measured fluorescence emission of Laurdan in phospholipid vesicles of 30, 50, and 100 nm diameter to show that osmotically induced membrane tension leads to an increase in polarity (hydration depth) of the phospholipid bilayer interior. Our data indicate that the general polarization of Laurdan emission is linearly dependent on membrane tension. We also show that higher membrane curvature leads to higher hydration levels. We anticipate that the proposed method will facilitate future studies of mechanically induced changes in physical properties of lipid bilayer environment both in vitro and in vivo.

  3. DNA Release from Lipoplexes by Anionic Lipids: Correlation with Lipid Mesomorphism, Interfacial Curvature, and Membrane Fusion

    PubMed Central

    Tarahovsky, Yury S.; Koynova, Rumiana; MacDonald, Robert C.

    2004-01-01

    DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells. PMID:15298910

  4. OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites.

    PubMed

    Kentala, Henriikka; Weber-Boyvat, Marion; Olkkonen, Vesa M

    2016-01-01

    Oxysterol-binding protein (OSBP) and its related protein homologs, ORPs, constitute a conserved family of lipid-binding/transfer proteins (LTPs) expressed ubiquitously in eukaryotes. The ligand-binding domain of ORPs accommodates cholesterol and oxysterols, but also glycerophospholipids, particularly phosphatidylinositol-4-phosphate (PI4P). ORPs have been implicated as intracellular lipid sensors or transporters. Most ORPs carry targeting determinants for the endoplasmic reticulum (ER) and non-ER organelle membrane. ORPs are located and function at membrane contact sites (MCSs), at which ER is closely apposed with other organelle limiting membranes. Such sites have roles in lipid transport and metabolism, control of Ca(2+) fluxes, and signaling events. ORPs are postulated either to transport lipids over MCSs to maintain the distinct lipid compositions of organelle membranes, or to control the activity of enzymes/protein complexes with functions in signaling and lipid metabolism. ORPs may transfer PI4P and another lipid class bidirectionally. Transport of PI4P followed by its hydrolysis would in this model provide the energy for transfer of the other lipid against its concentration gradient. Control of organelle lipid compositions by OSBP/ORPs is important for the life cycles of several pathogenic viruses. Targeting ORPs with small-molecular antagonists is proposed as a new strategy to combat viral infections. Several ORPs are reported to modulate vesicle transport along the secretory or endocytic pathways. Moreover, antagonists of certain ORPs inhibit cancer cell proliferation. Thus, ORPs are LTPs, which mediate interorganelle lipid transport and coordinate lipid signals with a variety of cellular regimes.

  5. Experimental Observations of Dynamic Critical Phenomena in a Lipid Membrane

    NASA Astrophysics Data System (ADS)

    Honerkamp-Smith, Aurelia R.; Machta, Benjamin B.; Keller, Sarah L.

    2012-06-01

    Near a critical point, the time scale of thermally induced fluctuations diverges in a manner determined by the dynamic universality class. Experiments have verified predicted three-dimensional dynamic critical exponents in many systems, but similar experiments in two dimensions have been lacking for the case of conserved order parameter. Here we analyze the time-dependent correlation functions of a quasi-two-dimensional lipid bilayer in water to show that its critical dynamics agree with a recently predicted universality class. In particular, the effective dynamic exponent zeff crosses over from ˜2 to ˜3 as the correlation length of fluctuations exceeds a hydrodynamic length set by the membrane and bulk viscosities.

  6. Lipid membranes in external electric fields: kinetics of large pore formation causing rupture.

    PubMed

    Winterhalter, Mathias

    2014-06-01

    About 40 years ago, Helfrich introduced an elastic model to explain shapes and shape transitions of cells (Z Naturforsch C, 1973; 28:693). This seminal article stimulated numerous theoretical as well as experimental investigations and created new research fields. In particular, the predictive power of his approach was demonstrated in a large variety of lipid model system. Here in this review, we focus on the development with respect to planar lipid membranes in external electric fields. Stimulated by the early work of Helfrich on electric field forces acting on liposomes, we extended his early approach to understand the kinetics of lipid membrane rupture. First, we revisit the main forces determining the kinetics of membrane rupture followed by an overview on various experiments. Knowledge on the kinetics of defect formation may help to design stable membranes or serve for novel mechanism for controlled release.

  7. Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output.

    PubMed

    Zhou, Yong; Prakash, Priyanka; Liang, Hong; Cho, Kwang-Jin; Gorfe, Alemayehu A; Hancock, John F

    2017-01-12

    K-Ras is targeted to the plasma membrane by a C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain. We used quantitative spatial imaging and atomistic molecular dynamics simulations to examine molecular details of K-Ras plasma membrane binding. We found that the K-Ras anchor binds selected plasma membrane anionic lipids with defined head groups and lipid side chains. The precise amino acid sequence and prenyl group define a combinatorial code for lipid binding that extends beyond simple electrostatics; within this code lysine and arginine residues are non-equivalent and prenyl chain length modifies nascent polybasic domain lipid preferences. The code is realized by distinct dynamic tertiary structures of the anchor on the plasma membrane that govern amino acid side-chain-lipid interactions. An important consequence of this specificity is the ability of such anchors when aggregated to sort subsets of phospholipids into nanoclusters with defined lipid compositions that determine K-Ras signaling output.

  8. EDTA-induced membrane fluidization and destabilization: biophysical studies on artificial lipid membranes.

    PubMed

    Prachayasittikul, Virapong; Isarankura-Na-Ayudhya, Chartchalerm; Tantimongcolwat, Tanawut; Nantasenamat, Chanin; Galla, Hans-Joachim

    2007-11-01

    The molecular mechanism of ethylenediaminetetraacetic acid (EDTA)-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixtures of DPPC and metal-chelating lipids, such as N(alpha),N(alpha)-Bis[carboxymethyl]-N(epsilon)-[(dioctadecylamino)succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.

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

    PubMed Central

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

    2014-01-01

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

  10. Probing protein-lipid interactions by FRET between membrane fluorophores

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

    2016-09-01

    Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

  11. Lipid Bilayer Vesicles with Numbers of Membrane-Linking Pores

    NASA Astrophysics Data System (ADS)

    Ken-ichirou Akashi,; Hidetake Miyata,

    2010-06-01

    We report that phospholipid membranes spontaneously formed in aqueous medium giant unilamellar vesicles (GUVs) possessing many membranous wormhole-like structures (membrane-linking pores, MLPs). By phase contract microscopy and confocal fluorescence microscopy, the structures of the MLPs, consisting of lipid bilayer, were resolvable, and a variety of vesicular shapes having many MLPs (a high genus topology) were found. These vesicles were stable but easily deformed by micromanipulation with a microneedle. We also observed the size reduction of the MLPs with the increase in membrane tension, which was qualitatively consistent with a prediction from a simple dynamical model.

  12. Phosphoinositides, Major Actors in Membrane Trafficking and Lipid Signaling Pathways

    PubMed Central

    De Craene, Johan-Owen; Bertazzi, Dimitri L.; Bär, Séverine; Friant, Sylvie

    2017-01-01

    Phosphoinositides are lipids involved in the vesicular transport of proteins and lipids between the different compartments of eukaryotic cells. They act by recruiting and/or activating effector proteins and thus are involved in regulating various cellular functions, such as vesicular budding, membrane fusion and cytoskeleton dynamics. Although detected in small concentrations in membranes, their role is essential to cell function, since imbalance in their concentrations is a hallmark of many cancers. Their synthesis involves phosphorylating/dephosphorylating positions D3, D4 and/or D5 of their inositol ring by specific lipid kinases and phosphatases. This process is tightly regulated and specific to the different intracellular membranes. Most enzymes involved in phosphoinositide synthesis are conserved between yeast and human, and their loss of function leads to severe diseases (cancer, myopathy, neuropathy and ciliopathy). PMID:28294977

  13. Molecular mechanisms of protein and lipid targeting to ciliary membranes

    PubMed Central

    Emmer, Brian T.; Maric, Danijela; Engman, David M.

    2010-01-01

    Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle. PMID:20145001

  14. Super-resolution optical microscopy of lipid plasma membrane dynamics.

    PubMed

    Eggeling, Christian

    2015-01-01

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory.

  15. Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

    PubMed Central

    Widomska, Justyna; Raguz, Marija; Subczynski, Witold K.

    2007-01-01

    The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. Epub 2007 March 20). At 35°C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties. PMID:17662231

  16. Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

    PubMed Central

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  17. Lateral diffusion of membrane proteins: consequences of hydrophobic mismatch and lipid composition.

    PubMed

    Ramadurai, Sivaramakrishnan; Duurkens, Ria; Krasnikov, Victor V; Poolman, Bert

    2010-09-08

    Biological membranes are composed of a large number lipid species differing in hydrophobic length, degree of saturation, and charge and size of the headgroup. We now present data on the effect of hydrocarbon chain length of the lipids and headgroup composition on the lateral mobility of the proteins in model membranes. The trimeric glutamate transporter (GltT) and the monomeric lactose transporter (LacY) were reconstituted in giant unilamellar vesicles composed of unsaturated phosphocholine lipids of varying acyl chain length (14-22 carbon atoms) and various ratios of DOPE/DOPG/DOPC lipids. The lateral mobility of the proteins and of a fluorescent lipid analog was determined as a function of the hydrophobic thickness of the bilayer (h) and lipid composition, using fluorescence correlation spectroscopy. The diffusion coefficient of LacY decreased with increasing thickness of the bilayer, in accordance with the continuum hydrodynamic model of Saffman-Delbrück. For GltT, the mobility had its maximum at diC18:1 PC, which is close to the hydrophobic thickness of the bilayer in vivo. The lateral mobility decreased linearly with the concentration of DOPE but was not affected by the fraction of anionic lipids from DOPG. The addition of DOPG and DOPE did not affect the activity of GltT. We conclude that the hydrophobic thickness of the bilayer is a major determinant of molecule diffusion in membranes, but protein-specific properties may lead to deviations from the Saffman-Delbrück model.

  18. Dynamic Response of Model Lipid Membranes to Ultrasonic Radiation Force

    PubMed Central

    Prieto, Martin Loynaz; Oralkan, Ömer; Khuri-Yakub, Butrus T.; Maduke, Merritt C.

    2013-01-01

    Low-intensity ultrasound can modulate action potential firing in neurons in vitro and in vivo. It has been suggested that this effect is mediated by mechanical interactions of ultrasound with neural cell membranes. We investigated whether these proposed interactions could be reproduced for further study in a synthetic lipid bilayer system. We measured the response of protein-free model membranes to low-intensity ultrasound using electrophysiology and laser Doppler vibrometry. We find that ultrasonic radiation force causes oscillation and displacement of lipid membranes, resulting in small (<1%) changes in membrane area and capacitance. Under voltage-clamp, the changes in capacitance manifest as capacitive currents with an exponentially decaying sinusoidal time course. The membrane oscillation can be modeled as a fluid dynamic response to a step change in pressure caused by ultrasonic radiation force, which disrupts the balance of forces between bilayer tension and hydrostatic pressure. We also investigated the origin of the radiation force acting on the bilayer. Part of the radiation force results from the reflection of the ultrasound from the solution/air interface above the bilayer (an effect that is specific to our experimental configuration) but part appears to reflect a direct interaction of ultrasound with the bilayer, related to either acoustic streaming or scattering of sound by the bilayer. Based on these results, we conclude that synthetic lipid bilayers can be used to study the effects of ultrasound on cell membranes and membrane proteins. PMID:24194863

  19. Lamellar biogels: Fluid-membrane-based hydrogels containing polymer lipids

    SciTech Connect

    Warriner, H.E.; Idziak, S.H.J.; Slack, N.L.

    1996-02-16

    A class of lamellar biological hydrogels comprised of fluid membranes of lipids and surfactants with small amounts of low molecular weight poly(ethylene glycol)-derived polymer pipids (PEG-lipids) were studied by x-ray diffraction, polarized light microscopy, and rheometry. In contrast to isotropic hydrogels of polymer networks, these membrane-based birefringent liquid crystalline biogels, labeled L{sub {alpha},g,} form the gel phase when water is added to the liquid-like lamellar L{sub {alpha}} phase, which reenters a liquid-like mixed phase upon further dilution. Furthermore, gels with larger water content require less PEG-lipid to remain stable. Although concentrated ({approx}50 weight percent) mixtures of free PEG (molecular weight, 5000) and water do not gel, gelatin does occur in mixtures containing as little as 0.5 weight percent PEG lipid. A defining signature of the L{sub {alpha}, g} regime as it sets in from the fluid lamellar L{sub {alpha}} phase is the proliferation of layer-dislocation-type defects, which are stabilized by the segregation of PEG-lipids to the defect regions of high membrane curvature that connect the membranes. 32 refs., 5 figs.

  20. Membrane Lipid Metabolism in Germinating Castor Bean Endosperm 1

    PubMed Central

    Donaldson, Robert P.

    1976-01-01

    Castor bean (Ricinus communis L. var. Hale) endosperms, excised after 2 days germination at 30 C, were incubated 5 min to 8 hr with 14C-acetate and 3H-glycerol. Homogenates were fractionated by sucrose gradient centrifugation. Organelles found to be active in lipid synthesis were the lipid bodies and the endoplasmic reticulum. The products of incorporation in the lipid bodies were 3H-diglycerides containing 14C-fatty acids of more than 20 carbons. In contrast, the endoplasmic reticulum produced 3H-phospholipids as well as 3H-diglycerides rich in 14C-linoleate. The phospholipids synthesized and their acyl contents were of the types known to be the major components of organelle membranes in this tissue. Phospholipids and diglycerides containing 14C and 3H were found in the glyoxysomes and mitochondria subsequent to their appearance in the endoplasmic reticulum. The results show that germinating castor bean endosperm synthesizes membrane lipids de novo from acetate rather than reutilizing stored lipid components directly. It is also apparent that the endoplasmic reticulum is responsible for several steps in membrane lipid production. PMID:16659516

  1. Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

    PubMed Central

    Jones, Emmalee M.; Dubey, Manish; Camp, Phillip J.; Vernon, Briana C.; Biernat, Jacek; Mandelkow, Eckhard; Majewski, Jaroslaw; Chi, Eva Y.

    2012-01-01

    The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that lipid membrane can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40's presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40's strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases. PMID:22401494

  2. Fatty Acids from Membrane Lipids Become Incorporated into Lipid Bodies during Myxococcus xanthus Differentiation

    PubMed Central

    Bhat, Swapna; Boynton, Tye O.; Pham, Dan; Shimkets, Lawrence J.

    2014-01-01

    Myxococcus xanthus responds to amino acid limitation by producing fruiting bodies containing dormant spores. During development, cells produce triacylglycerides in lipid bodies that become consumed during spore maturation. As the cells are starved to induce development, the production of triglycerides represents a counterintuitive metabolic switch. In this paper, lipid bodies were quantified in wild-type strain DK1622 and 33 developmental mutants at the cellular level by measuring the cross sectional area of the cell stained with the lipophilic dye Nile red. We provide five lines of evidence that triacylglycerides are derived from membrane phospholipids as cells shorten in length and then differentiate into myxospores. First, in wild type cells, lipid bodies appear early in development and their size increases concurrent with an 87% decline in membrane surface area. Second, developmental mutants blocked at different stages of shortening and differentiation accumulated lipid bodies proportionate with their cell length with a Pearson's correlation coefficient of 0.76. Third, peripheral rods, developing cells that do not produce lipid bodies, fail to shorten. Fourth, genes for fatty acid synthesis are down-regulated while genes for fatty acid degradation are up regulated. Finally, direct movement of fatty acids from membrane lipids in growing cells to lipid bodies in developing cells was observed by pulse labeling cells with palmitate. Recycling of lipids released by Programmed Cell Death appears not to be necessary for lipid body production as a fadL mutant was defective in fatty acid uptake but proficient in lipid body production. The lipid body regulon involves many developmental genes that are not specifically involved in fatty acid synthesis or degradation. MazF RNA interferase and its target, enhancer-binding protein Nla6, appear to negatively regulate cell shortening and TAG accumulation whereas most cell-cell signals activate these processes. PMID:24906161

  3. Hybrid lipids increase nanoscale fluctuation lifetimes in mixed membranes

    NASA Astrophysics Data System (ADS)

    Palmieri, Benoit; Safran, Samuel A.

    2013-09-01

    A recently proposed ternary mixture model is used to predict fluctuation domain lifetimes in the one phase region. The membrane is made of saturated, unsaturated, and hybrid lipids that have one saturated and one unsaturated hydrocarbon chain. The hybrid lipid is a natural linactant which can reduce the packing incompatibility between saturated and unsaturated lipids. The fluctuation lifetimes are predicted as a function of the hybrid lipid fraction and the fluctuation domain size. These lifetimes can be increased by up to three orders of magnitude compared to the case of no hybrids. With hybrid, small length scale fluctuations have sizable amplitudes even close to the critical temperature and, hence, benefit from enhanced critical slowing down. The increase in lifetime is particularly important for nanometer scale fluctuation domains where the hybrid orientation and the other lipids composition are highly coupled.

  4. Hybrid lipids increase nanoscale fluctuation lifetimes in mixed membranes.

    PubMed

    Palmieri, Benoit; Safran, Samuel A

    2013-09-01

    A recently proposed ternary mixture model is used to predict fluctuation domain lifetimes in the one phase region. The membrane is made of saturated, unsaturated, and hybrid lipids that have one saturated and one unsaturated hydrocarbon chain. The hybrid lipid is a natural linactant which can reduce the packing incompatibility between saturated and unsaturated lipids. The fluctuation lifetimes are predicted as a function of the hybrid lipid fraction and the fluctuation domain size. These lifetimes can be increased by up to three orders of magnitude compared to the case of no hybrids. With hybrid, small length scale fluctuations have sizable amplitudes even close to the critical temperature and, hence, benefit from enhanced critical slowing down. The increase in lifetime is particularly important for nanometer scale fluctuation domains where the hybrid orientation and the other lipids composition are highly coupled.

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

    PubMed Central

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

    2007-01-01

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

  6. Quantitative analysis of molecular partition towards lipid membranes using surface plasmon resonance

    PubMed Central

    Figueira, Tiago N.; Freire, João M.; Cunha-Santos, Catarina; Heras, Montserrat; Gonçalves, João; Moscona, Anne; Porotto, Matteo; Salomé Veiga, Ana; Castanho, Miguel A. R. B.

    2017-01-01

    Understanding the interplay between molecules and lipid membranes is fundamental when studying cellular and biotechnological phenomena. Partition between aqueous media and lipid membranes is key to the mechanism of action of many biomolecules and drugs. Quantifying membrane partition, through adequate and robust parameters, is thus essential. Surface Plasmon Resonance (SPR) is a powerful technique for studying 1:1 stoichiometric interactions but has limited application to lipid membrane partition data. We have developed and applied a novel mathematical model for SPR data treatment that enables determination of kinetic and equilibrium partition constants. The method uses two complementary fitting models for association and dissociation sensorgram data. The SPR partition data obtained for the antibody fragment F63, the HIV fusion inhibitor enfuvirtide, and the endogenous drug kyotorphin towards POPC membranes were compared against data from independent techniques. The comprehensive kinetic and partition models were applied to the membrane interaction data of HRC4, a measles virus entry inhibitor peptide, revealing its increased affinity for, and retention in, cholesterol-rich membranes. Overall, our work extends the application of SPR beyond the realm of 1:1 stoichiometric ligand-receptor binding into a new and immense field of applications: the interaction of solutes such as biomolecules and drugs with lipids. PMID:28358389

  7. Quantitative analysis of molecular partition towards lipid membranes using surface plasmon resonance.

    PubMed

    Figueira, Tiago N; Freire, João M; Cunha-Santos, Catarina; Heras, Montserrat; Gonçalves, João; Moscona, Anne; Porotto, Matteo; Salomé Veiga, Ana; Castanho, Miguel A R B

    2017-03-30

    Understanding the interplay between molecules and lipid membranes is fundamental when studying cellular and biotechnological phenomena. Partition between aqueous media and lipid membranes is key to the mechanism of action of many biomolecules and drugs. Quantifying membrane partition, through adequate and robust parameters, is thus essential. Surface Plasmon Resonance (SPR) is a powerful technique for studying 1:1 stoichiometric interactions but has limited application to lipid membrane partition data. We have developed and applied a novel mathematical model for SPR data treatment that enables determination of kinetic and equilibrium partition constants. The method uses two complementary fitting models for association and dissociation sensorgram data. The SPR partition data obtained for the antibody fragment F63, the HIV fusion inhibitor enfuvirtide, and the endogenous drug kyotorphin towards POPC membranes were compared against data from independent techniques. The comprehensive kinetic and partition models were applied to the membrane interaction data of HRC4, a measles virus entry inhibitor peptide, revealing its increased affinity for, and retention in, cholesterol-rich membranes. Overall, our work extends the application of SPR beyond the realm of 1:1 stoichiometric ligand-receptor binding into a new and immense field of applications: the interaction of solutes such as biomolecules and drugs with lipids.

  8. A rapid method for determining arachidonic:eicosapentaenoic acid ratios in whole blood lipids: correlation with erythrocyte membrane ratios and validation in a large Italian population of various ages and pathologies

    PubMed Central

    2010-01-01

    Background Omega-3 and -6 polyunsaturated fatty acids (LCPUFA), are important for good health conditions. They are present in membrane phospholipids. The ratio of total n-6:n-3 LCPUFA and arachidonic acid:eicosapentaenoic acid (AA and EPA), should not exceed 5:1. Increased intake of n-6 and decreased consumption of n-3 has resulted in much higher, ca 10/15:1 ratio in RBC fatty acids with the possible appearance of a pathological "scenario". The determination of RBC phospholipid LCPUFA contents and ratios is the method of choice for assessing fatty acid status but it is labour intensive and time consuming. Aims of the study [i] To describe and validate a rapid method, suitable for large scale population studies, for total blood fatty acid assay; [ii] to verify a possible correlation between total n-6:n-3 ratio and AA:EPA ratios in RBC phospholipids and in whole-blood total lipids, [iii] to assess usefulness of these ratio as biomarkers of LCPUFA status. Methods [1] Healthy volunteers and patients with various pathologies were recruited. [2] Fatty acid analyses by GC of methyl esters from directly derivatized whole blood total lipids and from RBC phospholipids were performed on fasting blood samples from 1432 subjects categorised according to their age, sex and any existing pathologies. AA:EPA ratio and the total n-6:n-3 ratio were determined. Results AA:EPA ratio is a more sensitive and reliable index for determining changes in total blood fatty acid and it is correlated with the ratio derived from extracted RBC phospholipids. Conclusions The described AA:EPA ratio is a simple, rapid and reliable method for determining n-3 fatty acid status. PMID:20105293

  9. Stabilization of Lipid Membranes With Dendritic Polymers

    DTIC Science & Technology

    2004-12-01

    monolayer coverage was possible using the spin - coating procedure for concentrations of 10-5 w/w dendrimers in solution or lower. Higher coverages...The fluorescence intensity increased as coverage time before spin coating increased and as the dendrimer solution concentration increased (Fig...enhanced the adsorption of lipids to the substrate. Figure 7: Fluorescence intensity as a function of the PAMAM concentration used in the spin

  10. Computational redesign of the lipid-facing surface of the outer membrane protein OmpA.

    PubMed

    Stapleton, James A; Whitehead, Timothy A; Nanda, Vikas

    2015-08-04

    Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

  11. Interaction of pristine and functionalized carbon nanotubes with lipid membranes.

    PubMed

    Baoukina, Svetlana; Monticelli, Luca; Tieleman, D Peter

    2013-10-10

    Carbon nanotubes are widely used in a growing number of applications. Their interactions with biological materials, cell membranes in particular, is of interest in applications including drug delivery and for understanding the toxicity of carbon nanotubes. We use extensive molecular dynamics simulations with the MARTINI model to study the interactions of model nanotubes of different thickness, length, and patterns of chemical modification with model membranes. In addition, we characterize the interactions of small bundles of carbon nanotubes with membrane models. Short pristine carbon nanotubes readily insert into membranes and adopt an orientation parallel to the plane of the membrane in the center of the membrane. Larger aggregates and functionalized nanotubes exhibit a range of possible interactions. The distribution and orientation of carbon nanotubes can be controlled by functionalizing the nanotubes. Free energy calculations provide thermodynamic insight into the preferred orientations of different nanotubes and quantify structural defects in the lipid matrix.

  12. Reversible control of current across lipid membranes by local heating

    PubMed Central

    Urban, Patrick; Kirchner, Silke R.; Mühlbauer, Christian; Lohmüller, Theobald; Feldmann, Jochen

    2016-01-01

    Lipid membranes are almost impermeable for charged molecules and ions that can pass the membrane barrier only with the help of specialized transport proteins. Here, we report how temperature manipulation at the nanoscale can be employed to reversibly control the electrical resistance and the amount of current that flows through a bilayer membrane with pA resolution. For this experiment, heating is achieved by irradiating gold nanoparticles that are attached to the bilayer membrane with laser light at their plasmon resonance frequency. We found that controlling the temperature on the nanoscale renders it possible to reproducibly regulate the current across a phospholipid membrane and the membrane of living cells in absence of any ion channels. PMID:26940847

  13. Cholesterol effect on the dipole potential of lipid membranes.

    PubMed

    Starke-Peterkovic, Thomas; Turner, Nigel; Vitha, Mark F; Waller, Mark P; Hibbs, David E; Clarke, Ronald J

    2006-06-01

    The effect of cholesterol removal by methyl-beta-cyclodextrin on the dipole potential, psi(d), of membrane vesicles composed of natural membrane lipids extracted from the kidney and brain of eight vertebrate species was investigated using the voltage-sensitive fluorescent probe di-8-ANEPPS. Cyclodextrin treatment reduced cholesterol levels by on average 80% and this was associated with an average reduction in psi(d) of 50 mV. Measurements of the effect of a range of cholesterol derivatives on the psi(d) of DMPC lipid vesicles showed that the magnitude of the effect correlated with the component of the sterol's dipole moment perpendicular to the membrane surface. The changes in psi(d) observed could not be accounted for solely by the electric field originating from the sterols' dipole moments. Additional factors must arise from sterol-induced changes in lipid packing, which changes the density of dipoles in the membrane, and changes in water penetration into the membrane, which changes the effective dielectric constant of the interfacial region. In DMPC membranes, the cholesterol-induced change in psi(d) was biphasic, i.e., a maximum in psi(d) was observed at approximately 35-45 mol %, after which psi(d) started to decrease. We suggest that this could be associated with a maximum in the strength of DMPC-cholesterol intermolecular forces at this composition.

  14. Analysis of the Torus Surrounding Planar Lipid Bilayer Membranes

    PubMed Central

    White, Stephen H.

    1972-01-01

    The characteristics and behavior of the torus (annulus) surrounding planar lipid bilayer membranes formed across a cylindrical aperture are analyzed using equations for the shape and volume of the annulus derived by the methods of variational calculus. The analysis leads to the following results: (a) Design criteria for the aperture can be established. (b) The transition region between thin film and thick annulus can be defined quantitatively and its effect on the measurement of specific capacitance determined. (c) At fixed annulus volume the diameter of the thin membrane is a function of the thin film-annulus contact angle. This suggests a new method for examining changes in free energy of the thin film, and explains why the area of thin film increases reversibly when potentials are present across the film. (d) In the absence of buoyant forces, the equations for the shape and volume of the annulus consist of incomplete elliptic integrals of the first and second kinds; however, the shape of the annulus in the transition region can be described with good accuracy by an approximate equation of greater simplicity. PMID:5019479

  15. Analysis of the torus surrounding planar lipid bilayer membranes.

    PubMed

    White, S H

    1972-04-01

    The characteristics and behavior of the torus (annulus) surrounding planar lipid bilayer membranes formed across a cylindrical aperture are analyzed using equations for the shape and volume of the annulus derived by the methods of variational calculus. The analysis leads to the following results: (a) Design criteria for the aperture can be established. (b) The transition region between thin film and thick annulus can be defined quantitatively and its effect on the measurement of specific capacitance determined. (c) At fixed annulus volume the diameter of the thin membrane is a function of the thin film-annulus contact angle. This suggests a new method for examining changes in free energy of the thin film, and explains why the area of thin film increases reversibly when potentials are present across the film. (d) In the absence of buoyant forces, the equations for the shape and volume of the annulus consist of incomplete elliptic integrals of the first and second kinds; however, the shape of the annulus in the transition region can be described with good accuracy by an approximate equation of greater simplicity.

  16. Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes.

    PubMed

    Dubois, Louise; Ronquist, Karl K Göran; Ek, Bo; Ronquist, Gunnar; Larsson, Anders

    2015-11-01

    Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

  17. 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

  18. Photon correlation spectroscopy of bilayer lipid membranes.

    PubMed Central

    Crilly, J F; Earnshaw, J C

    1983-01-01

    Light scattering by thermal fluctuations on simple monoglyceride bilayer membranes has been used to investigate the viscoelastic properties of these structures. Spectroscopic analysis of these fluctuations (capillary waves) permits the nonperturbative measurement of the interfacial tension and a shear interfacial viscosity acting normal to the membrane plane. The methods were established by studies of solvent and nonsolvent bilayers of glycerol monooleate (GMO). Changes in the tension of GMO/n-decane membranes induced by altering the composition of the parent solution were detected and quantified. In a test of the reliability of the technique controlled variations of the viscosity of the aqueous bathing solution were accurately monitored. The technique was applied to solvent-free bilayers formed from dispersions of GMO in squalane. The lower tensions observed attested to the comparative absence of solvent in such bilayers. In contrast to the solvent case, the solvent-free membranes exhibited a significant transverse shear viscosity, indicative of the enhanced intermolecular interactions within the bilayer. PMID:6838962

  19. Stabilization of composition fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Safran, Samuel; Palmieri, Benoit

    2013-03-01

    A ternary mixture model is proposed to describe composition fluctuations in mixed membranes composed of saturated, unsaturated and hybrid lipids. The asymmetric hybrid lipid has one saturated and one unsaturated hydrocarbon chain and it can reduce the packing incompatibility between saturated and unsaturated lipids. A methodology to recast the free-energy of the lattice in terms of a continuous isotropic field theory is proposed and used to analyze composition fluctuations above the critical temperature. The effect of hybrid lipids on fluctuations domains rich in saturated/unsaturated lipids is predicted. The correlation length of such fluctuations decreases significantly with increasing amounts of hybrids even if the temperature is maintained close to the critical temperature. This provides an upper bound for the domain sizes expected in rafts stabilized by hybrids, above the critical temperature. When the hybrid composition of the membrane is increased further, a crossover value is found above which ``stripe-like'' fluctuations are observed. The wavelength of these fluctuations decreases with increasing hybrid fraction and tends toward a molecular size in a membrane that contains only hybrids.

  20. Reduced Lateral Mobility of Lipids and Proteins in Crowded Membranes

    PubMed Central

    Goose, Joseph E.; Sansom, Mark S. P.

    2013-01-01

    Coarse-grained molecular dynamics simulations of the E. coli outer membrane proteins FhuA, LamB, NanC, OmpA and OmpF in a POPE/POPG (3∶1) bilayer were performed to characterise the diffusive nature of each component of the membrane. At small observation times (<10 ns) particle vibrations dominate phospholipid diffusion elevating the calculated values from the longer time-scale bulk value (>50 ns) of 8.5×10−7 cm2 s−1. The phospholipid diffusion around each protein was found to vary based on distance from protein. An asymmetry in the diffusion of annular lipids in the inner and outer leaflets was observed and correlated with an asymmetry in charged residues in the vicinity of the inner and outer leaflet head-groups. Protein rotational and translational diffusion were also found to vary with observation time and were inversely correlated with the radius of gyration of the protein in the plane of the bilayer. As the concentration of protein within the bilayer was increased, the overall mobility of the membrane decreased reflected in reduced lipid diffusion coefficients for both lipid and protein components. The increase in protein concentration also resulted in a decrease in the anomalous diffusion exponent α of the lipid. Formation of extended clusters and networks of proteins led to compartmentalisation of lipids in extreme cases. PMID:23592975

  1. Anesthetic diffusion through lipid membranes depends on the protonation rate.

    PubMed

    Pérez-Isidoro, Rosendo; Sierra-Valdez, F J; Ruiz-Suárez, J C

    2014-12-18

    Hundreds of substances possess anesthetic action. However, despite decades of research and tests, a golden rule is required to reconcile the diverse hypothesis behind anesthesia. What makes an anesthetic to be local or general in the first place? The specific targets on proteins, the solubility in lipids, the diffusivity, potency, action time? Here we show that there could be a new player equally or even more important to disentangle the riddle: the protonation rate. Indeed, such rate modulates the diffusion speed of anesthetics into lipid membranes; low protonation rates enhance the diffusion for local anesthetics while high ones reduce it. We show also that there is a pH and membrane phase dependence on the local anesthetic diffusion across multiple lipid bilayers. Based on our findings we incorporate a new clue that may advance our understanding of the anesthetic phenomenon.

  2. 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.

  3. 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

  4. Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum

    PubMed Central

    Takahashi, Kei; Toyota, Taro

    2017-01-01

    Background: 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. PMID:28272354

  5. Methodologies for the Analysis of Instantaneous Lipid Diffusion in MD Simulations of Large Membrane Systems

    PubMed Central

    Chavent, Matthieu; Reddy, Tyler; Goose, Joseph; Dahl, Anna Caroline E.; Stone, John E.; Jobard, Bruno; Sansom, Mark S.P.

    2014-01-01

    Interactions between lipids and membrane proteins play a key role in determining the nanoscale dynamic and structural properties of biological membranes. Molecular dynamics (MD) simulations provide a valuable tool for studying membrane models, complementing experimental approaches. It is now possible to simulate large membrane systems, such as simplified models of bacterial and viral envelope membranes. Consequently, there is a pressing need to develop tools to visualize and quantify the dynamics of these immense systems, which typically are comprised of millions of particles. To tackle this issue, we have developed visual and quantitative analyses of molecular positions and their velocity field using path line, vector field and streamline techniques. This allows us to highlight large, transient flow-like movements of lipids and to better understand crowding within the lipid bilayer. The current study focuses on visualization and analysis of lipid dynamics. However, the methods are flexible and can be readily applied to e.g. proteins and nanoparticles within large complex membranes. The protocols developed here are readily accessible both as a plugin for the molecular visualization program VMD and as a module for the MDAnalysis library. PMID:25341001

  6. Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel.

    PubMed

    Kolomiytseva, Iskra K; Perepelkina, Natalia I; Zharikova, Alevtina D; Popov, Victor I

    2008-12-01

    Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.

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

    PubMed

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

    2014-02-25

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

  8. Femtosecond crystallography of membrane proteins in the lipidic cubic phase

    PubMed Central

    Liu, Wei; Wacker, Daniel; Wang, Chong; Abola, Enrique; Cherezov, Vadim

    2014-01-01

    Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins. PMID:24914147

  9. Water penetration profile at the protein-lipid interface in Na,K-ATPase membranes.

    PubMed

    Bartucci, Rosa; Guzzi, Rita; Esmann, Mikael; Marsh, Derek

    2014-09-16

    The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from (2)H-hyperfine interaction with D2O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D2O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D2O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D2O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D2O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.

  10. Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea-Inspired Tetraether Lipids.

    PubMed

    Koyanagi, Takaoki; Leriche, Geoffray; Onofrei, David; Holland, Gregory P; Mayer, Michael; Yang, Jerry

    2016-01-26

    Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies.

  11. DNA-Tile Structures Induce Ionic Currents through Lipid Membranes.

    PubMed

    Göpfrich, Kerstin; Zettl, Thomas; Meijering, Anna E C; Hernández-Ainsa, Silvia; Kocabey, Samet; Liedl, Tim; Keyser, Ulrich F

    2015-05-13

    Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biological ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.

  12. Polyunsaturation in lipid membranes: dynamic properties and lateral pressure profiles.

    PubMed

    Ollila, Samuli; Hyvönen, Marja T; Vattulainen, Ilpo

    2007-03-29

    We elucidate the influence of unsaturation on single-component membrane properties, focusing on their dynamical aspects and lateral pressure profiles across the membrane. To this end, we employ atomistic molecular dynamics simulations to study five different membrane systems with varying degrees of unsaturation, starting from saturated membranes and systematically increasing the level of unsaturation, ending up with a bilayer of phospholipids containing the docosahexaenoic acid. For an increasing level of unsaturation, we find considerable effects on dynamical properties, such as accelerated dynamics of the phosphocholine head groups and glycerol backbones and speeded up rotational dynamics of the lipid molecules. The lateral pressure profile is found to be altered by the degree of unsaturation. For an increasing number of double bonds, the peak in the middle of the bilayer decreases. This is compensated for by changes in the membrane-water interface region in terms of increasing peak heights of the lateral pressure profile. Implications of the findings are briefly discussed.

  13. Artificial black membranes from bipolar lipids of thermophilic Archaebacteria.

    PubMed Central

    Gliozzi, A; Rolandi, R; De Rosa, M; Gambacorta, A

    1982-01-01

    The membrane of thermophilic archaebacteria is characterized by the presence of unusual isoprenoid bipolar lipids. The molecular organization of these lipids is still a matter of study. Important information could come from forming artificial black membranes. Black films can be formed from n-alkane or squalene dispersions of bipolar lipids extracted from the membrane of Caldariella acidophila. Membrane formation occurred only above a critical temperature (approximately 70 degrees C) corresponding to the physiological one. At lower temperatures, special solvent systems (n-alkanes or squalene, butanol and n-alkanes or squalene, butanol chloroform) were required. To characterize the physical parameters of these membranes, conductance and capacitance measurements were performed. Conductance was in the range of 10(-8) - 10(-7) omega -1 cm -2 , where specific capacitance at T = 72 degrees C was Cs = 0.685 +/- 0.004 microF/cm2 and Cs = 0.658 +/- 0.08 microF/cm2, corresponding to a dielectric thickness of 27 and 29 A for squalene and dodecane dispersions, respectively. Capacitance was shown to vary as the square of membrane potential, as usual in lipid bilayers. Values of the proportionality constant alpha have been compared to those of solvent-containing and solvent-free bilayers. The behavior of capacitance as a function of temperature is also shown by lowering temperature; the occurrence of complex structural changes was indicated. All the experimental data suggest that the presence of solvent is very low. Two possible molecular configurations of the films are discussed. PMID:6800415

  14. Distribution of Fullerene Nanoparticles between Water and Solid Supported Lipid Membranes: Thermodynamics and Effects of Membrane Composition on Distribution.

    PubMed

    Ha, Yeonjeong; Katz, Lynn E; Liljestrand, Howard M

    2015-12-15

    The distribution coefficient (Klipw) of fullerene between solid supported lipid membranes (SSLMs) and water was examined using different lipid membrane compositions. Klipw of fullerene was significantly higher with a cationic lipid membrane compared to that with a zwitterionic or anionic lipid membrane, potentially due to the strong interactions between negative fullerene dispersions and positive lipid head groups. The higher Klipw for fullerene distribution to ternary lipid mixture membranes was attributed to an increase in the interfacial surface area of the lipid membrane resulting from phase separation. These results imply that lipid composition can be a critical factor that affects bioconcentration of fullerene. Distribution of fullerene into zwitterionic unsaturated lipid membranes was dominated by the entropy contribution (ΔS) and the process was endothermic (ΔH > 0). This result contrasts the partitioning thermodynamics of highly and moderately hydrophobic chemicals indicating that the lipid-water distribution mechanism of fullerene may be different from that of molecular level chemicals. Potential mechanisms for the distribution of fullerene that may explain these differences include adsorption on the lipid membrane surfaces and partitioning into the center of lipid membranes (i.e., absorption).

  15. Incorporation of large guest molecules into liposomes via chemical reactions in lipid membranes.

    PubMed

    Tsuchiya, Yuki; Sugikawa, Kouta; Ueda, Masafumi; Ikeda, Atsushi

    2017-02-22

    The incorporation of hydrophobic guest molecules into lipid membranes by the exchange of the guest molecule from a cyclodextrin (CDx) complex to a liposome is limited to guest molecules that can be included in CDxs. To solve this problem, large guest molecules were incorporated into liposomes by chemical reactions of guest molecules in lipid membranes. Stable lipid-membrane-incorporated fullerene derivatives with large substituent(s) were prepared by Diels-Alder reactions in lipid membranes.

  16. Mechanical properties that influence antimicrobial peptide activity in lipid membranes.

    PubMed

    Marín-Medina, Nathaly; Ramírez, Diego Alejandro; Trier, Steve; Leidy, Chad

    2016-12-01

    Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.

  17. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes.

  18. Tuning Membrane Thickness Fluctuations in Model Lipid Bilayers

    PubMed Central

    Ashkar, Rana; Nagao, Michihiro; Butler, Paul D.; Woodka, Andrea C.; Sen, Mani K.; Koga, Tadanori

    2015-01-01

    Membrane thickness fluctuations have been associated with a variety of critical membrane phenomena, such as cellular exchange, pore formation, and protein binding, which are intimately related to cell functionality and effective pharmaceuticals. Therefore, understanding how these fluctuations are controlled can remarkably impact medical applications involving selective macromolecule binding and efficient cellular drug intake. Interestingly, previous reports on single-component bilayers show almost identical thickness fluctuation patterns for all investigated lipid tail-lengths, with similar temperature-independent membrane thickness fluctuation amplitude in the fluid phase and a rapid suppression of fluctuations upon transition to the gel phase. Presumably, in vivo functions require a tunability of these parameters, suggesting that more complex model systems are necessary. In this study, we explore lipid tail-length mismatch as a regulator for membrane fluctuations. Unilamellar vesicles of an equimolar mixture of dimyristoylphosphatidylcholine and distearoylphosphatidylcholine molecules, with different tail-lengths and melting transition temperatures, are used as a model system for this next level of complexity. Indeed, this binary system exhibits a significant response of membrane dynamics to thermal variations. The system also suggests a decoupling of the amplitude and the relaxation time of the membrane thickness fluctuations, implying a potential for independent control of these two key parameters. PMID:26153707

  19. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    SciTech Connect

    Rozovsky, Sharon; Forstner, Martin B.; Sondermann, Holger; Groves, Jay T.

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

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

    PubMed Central

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

    1995-01-01

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

  1. Important roles for membrane lipids in haloarchaeal bioenergetics.

    PubMed

    Kellermann, Matthias Y; Yoshinaga, Marcos Y; Valentine, Raymond C; Wörmer, Lars; Valentine, David L

    2016-11-01

    Recent advances in lipidomic analysis in combination with various physiological experiments set the stage for deciphering the structure-function of haloarchaeal membrane lipids. Here we focused primarily on changes in lipid composition of Haloferax volcanii, but also performed a comparative analysis with four other haloarchaeal species (Halobacterium salinarum, Halorubrum lacusprofundi, Halorubrum sodomense and Haloplanus natans) all representing distinctive cell morphologies and behaviors (i.e., rod shape vs. pleomorphic behavior). Common to all five haloarchaea, our data reveal an extraordinary high level of menaquinone, reaching up to 72% of the total lipids. This ubiquity suggests that menaquinones may function beyond their ordinary role as electron and proton transporter, acting simultaneously as ion permeability barriers and as powerful shield against oxidative stress. In addition, we aimed at understanding the role of cations interacting with the characteristic negatively charged surface of haloarchaeal membranes. We propose for instance that by bridging the negative charges of adjacent anionic phospholipids, Mg(2+) acts as surrogate for cardiolipin, a molecule that is known to control curvature stress of membranes. This study further provides a bioenergetic perspective as to how haloarchaea evolved following oxygenation of Earth's atmosphere. The success of the aerobic lifestyle of haloarchaea includes multiple membrane-based strategies that successfully balance the need for a robust bilayer structure with the need for high rates of electron transport - collectively representing the molecular basis to inhabit hypersaline water bodies around the planet.

  2. Buffers affect the bending rigidity of model lipid membranes.

    PubMed

    Bouvrais, Hélène; Duelund, Lars; Ipsen, John H

    2014-01-14

    In biophysical and biochemical studies of lipid bilayers the influence of the used buffer is often ignored or assumed to be negligible on membrane structure, elasticity, or physical properties. However, we here present experimental evidence, through bending rigidity measurements performed on giant vesicles, of a more complex behavior, where the buffering molecules may considerably affect the bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on the bending modulus is observed in the presence of both salt and buffer molecules, which serves as a warning to experimentalists in the data interpretation of their studies, since typical lipid bilayer studies contain buffer and ion molecules.

  3. Diffusion of water and selected atoms in DMPC lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

    Molecular dynamics simulations have been used to determine the diffusion of water molecules as a function of their position in a fully hydrated freestanding 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) bilayer membrane at 303 K and 1 atm. The diffusion rate of water in a ˜10 Å thick layer just outside the membrane surface is reduced on average by a factor of ˜2 relative to bulk. For water molecules penetrating deeper into the membrane, there is an increasing reduction in the average diffusion rate with up to one order of magnitude decrease for those deepest in the membrane. A comparison with the diffusion rate of selected atoms in the lipid molecules shows that ˜6 water molecules per lipid molecule move on the same time scale as the lipids and may therefore be considered to be tightly bound to them. The quasielastic neutron scattering functions for water and selected atoms in the lipid molecule have been simulated and compared to observed quasielastic neutron scattering spectra from single-supported bilayer DMPC membranes.

  4. Influence of membrane lipid composition on flavonoid-membrane interactions: Implications on their biological activity.

    PubMed

    Selvaraj, Stalin; Krishnaswamy, Sridharan; Devashya, Venkappayya; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2015-04-01

    The membrane interactions and localization of flavonoids play a vital role in altering membrane-mediated cell signaling cascades as well as influence the pharmacological activities such as anti-tumour, anti-microbial and anti-oxidant properties of flavonoids. Various techniques have been used to investigate the membrane interaction of flavonoids. These include partition coefficient, fluorescence anisotropy, differential scanning calorimetry, NMR spectroscopy, electrophysiological methods and molecular dynamics simulations. Each technique will provide specific information about either alteration of membrane fluidity or localization of flavonoids within the lipid bilayer. Apart from the diverse techniques employed, the concentrations of flavonoids and lipid membrane composition employed in various studies reported in literature also are different and together these variables contribute to diverse findings that sometimes contradict each other. This review highlights different techniques employed to investigate the membrane interaction of flavonoids with special emphasis on erythrocyte model membrane systems and their significance in understanding the nature and extent of flavonoid-membrane interactions. We also attempt to correlate the membrane localization and alteration in membrane fluidity with the biological activities of flavonoids such as anti-oxidant, anti-cancer and anti-microbial properties.

  5. Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

    SciTech Connect

    Tenchov, Boris G; Wang, Li; Koynova, Rumiana; MacDonald, Robert C

    2010-01-18

    Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by {approx}10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L{sub {alpha}} {yields} H{sub II}, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L{sub {alpha}} {yields} H{sub II} phase transition and promoted formation of an inverted cubic phase between the L{sub {alpha}} and H{sub II} phases. In contrast, moderate and weak transfection agents retained the direct L{sub {alpha}} {yields} H{sub II} transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid

  6. Membrane lipid rafts and neurobiology: age-related changes in membrane lipids and loss of neuronal function.

    PubMed

    Egawa, Junji; Pearn, Matthew L; Lemkuil, Brian P; Patel, Piyush M; Head, Brian P

    2016-08-15

    A better understanding of the cellular physiological role that plasma membrane lipids, fatty acids and sterols play in various cellular systems may yield more insight into how cellular and whole organ function is altered during the ageing process. Membrane lipid rafts (MLRs) within the plasma membrane of most cells serve as key organizers of intracellular signalling and tethering points of cytoskeletal components. MLRs are plasmalemmal microdomains enriched in sphingolipids, cholesterol and scaffolding proteins; they serve as a platform for signal transduction, cytoskeletal organization and vesicular trafficking. Within MLRs are the scaffolding and cholesterol binding proteins named caveolin (Cav). Cavs not only organize a multitude of receptors including neurotransmitter receptors (NMDA and AMPA receptors), signalling proteins that regulate the production of cAMP (G protein-coupled receptors, adenylyl cyclases, phosphodiesterases (PDEs)), and receptor tyrosine kinases involved in growth (Trk), but also interact with components that modulate actin and tubulin cytoskeletal dynamics (e.g. RhoGTPases and actin binding proteins). MLRs are essential for the regulation of the physiology of organs such as the brain, and age-related loss of cholesterol from the plasma membrane leads to loss of MLRs, decreased presynaptic vesicle fusion, and changes in neurotransmitter release, all of which contribute to different forms of neurodegeneration. Thus, MLRs provide an active membrane domain that tethers and reorganizes the cytoskeletal machinery necessary for membrane and cellular repair, and genetic interventions that restore MLRs to normal cellular levels may be exploited as potential therapeutic means to reverse the ageing and neurodegenerative processes.

  7. [The action of week inorganic acids and lower carboxylic acids on the conductivity of bilayer lipid membranes].

    PubMed

    Kilivnik, K E; Khmarskaia, L A; Ksenzhek, O S

    2009-01-01

    The ability of weak inorganic acids (H2S, HCN) and lower carboxylic acids to interact with bilayer lipid membranes, change their conductivity, and act as protonophores has been investigated. The mechanism of changes in BLM conductivity was studied. Factors influencing the interaction of acids with model lipid membranes were determined. Maximum changes in conductivity were observed at pH values equal to the dissociation constants of weak acids and depend on the coefficients of distribution "octanol-water".

  8. Simulation of Nanoparticle Permeation through a Lipid Membrane

    PubMed Central

    Fiedler, Steven L.; Violi, Angela

    2010-01-01

    Abstract A metric of nanoparticle toxicity is the passive permeability rate through cellular membranes. To assess the influence of nanoparticle morphology on this process, the permeability of buckyball-sized molecules through a representative lipid bilayer was investigated by molecular-dynamics simulation. When C60 was compared with a prototypical opened C60 molecule and a representative combustion-generated particle, C68H29, the calculated free-energy profiles along the permeation coordinate revealed a sizable variation in form and depth. The orientation of the anisotropic molecules was determined by monitoring the principal axis corresponding to the largest moment of inertia, and free rotation was shown to be hindered in the bilayer interior. Diffusion constant values of the permeant molecules were calculated from a statistical average of seven to 10 trajectories at five locations along the permeation coordinate. A relatively minor variation of the values was observed in the bilayer interior; however, local resistance values spanned up to 24 orders of magnitude from the water layer to the bilayer center, due primarily to its exponential dependence on free energy. The permeability coefficient values calculated for the three similarly sized but structurally distinct nanoparticles showed a significant variance. The use of C60 to represent similarly sized carbonaceous nanoparticles for assessments of toxicity is questioned. PMID:20655842

  9. Physical aspects of heterogeneities in multi-component lipid membranes.

    PubMed

    Komura, Shigeyuki; Andelman, David

    2014-06-01

    Ever since the raft model for biomembranes has been proposed, the traditional view of biomembranes based on the fluid-mosaic model has been altered. In the raft model, dynamical heterogeneities in multi-component lipid bilayers play an essential role. Focusing on the lateral phase separation of biomembranes and vesicles, we review some of the most relevant research conducted over the last decade. We mainly refer to those experimental works that are based on physical chemistry approach, and to theoretical explanations given in terms of soft matter physics. In the first part, we describe the phase behavior and the conformation of multi-component lipid bilayers. After formulating the hydrodynamics of fluid membranes in the presence of the surrounding solvent, we discuss the domain growth-law and decay rate of concentration fluctuations. Finally, we review several attempts to describe membrane rafts as two-dimensional microemulsion.

  10. Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

    PubMed

    Orosz, Kristina S; Jones, Ian W; Keogh, John P; Smith, Christopher M; Griffin, Kaitlyn R; Xu, Juhua; Comi, Troy J; Hall, H K; Saavedra, S Scott

    2016-02-16

    Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

  11. Photopolymerization of dienoyl lipids creates planar supported poly(lipid) membranes with retained fluidity

    PubMed Central

    Orosz, Kristina S.; Jones, Ian W.; Keogh, John P.; Smith, Christopher M.; Griffin, Kaitlyn R.; Xu, Juhua; Comi, Troy J.; Hall, H. K.

    2016-01-01

    Polymerization of substrate-supported bilayers composed of dienoyl phosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability, however the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl phosphatidylcholine (mono-SorbPC), bis-dienoyl phosphatidylcholine (bis-DenPC) and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity, however measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate inter-leaflet bonding. The D values measured after polymerization were 0.1 to 0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases, and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed. PMID:26794208

  12. Analysis of membrane lipid biogenesis pathways using yeast genetics.

    PubMed

    Gsell, Martina; Daum, Günther

    2013-01-01

    The yeast Saccharomyces cerevisiae has become a valuable eukaryotic model organism to study biochemical and cellular processes at a molecular basis. A common strategy for such studies is the use of single and multiple mutants constructed by genetic manipulation which are compromised in individual enzymatic steps or certain metabolic pathways. Here, we describe selected examples of yeast research on phospholipid metabolism with emphasis on our own work dealing with investigations of phosphatidylethanolamine synthesis. Such studies start with the selection and construction of appropriate mutants and lead to phenotype analysis, lipid profiling, enzymatic analysis, and in vivo experiments. Comparing results obtained with wild-type and mutant strains allows us to understand the role of gene products and metabolic processes in more detail. Such studies are valuable not only for contributing to our knowledge of the complex network of lipid metabolism, but also of effects of lipids on structure and function of cellular membranes.

  13. Electrochemical characterization of bilayer lipid membrane-semiconductor junctions

    SciTech Connect

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

    1990-03-08

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

  14. Membrane Protein Crystallization in Lipidic Mesophases. Hosting lipid affects on the crystallization and structure of a transmembrane peptide.

    PubMed

    Höfer, Nicole; Aragão, David; Lyons, Joseph A; Caffrey, Martin

    2011-04-06

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation.

  15. Membrane Protein Crystallization in Lipidic Mesophases. Hosting lipid affects on the crystallization and structure of a transmembrane peptide

    PubMed Central

    Höfer, Nicole; Aragão, David; Lyons, Joseph A.; Caffrey, Martin

    2012-01-01

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation. PMID:22933857

  16. Membrane Protein Crystallization in Lipidic Mesophases. Hosting Lipid Effects on the Crystallization and Structure of a Transmembrane Peptide

    SciTech Connect

    Hfer, Nicole; Aragao, David; Lyons, Joseph A.; Caffrey, Martin

    2011-09-28

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating d- and l-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double-stranded, intertwined double helix (DSDH) or as a head-to-head single-stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis, the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesized lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high-quality, structure-grade crystals with gramicidin only in the DSDH conformation.

  17. Polymer-Induced Swelling of Solid-Supported Lipid Membranes

    PubMed Central

    Kreuzer, Martin; Trapp, Marcus; Dahint, Reiner; Steitz, Roland

    2015-01-01

    In this paper, we study the interaction of charged polymers with solid-supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes by in-situ neutron reflectivity. We observe an enormous swelling of the oligolamellar lipid bilayer stacks after incubation in solutions of poly(allylamine hydrochloride) (PAH) in D2O. The positively charged polyelectrolyte molecules interact with the lipid bilayers and induce a drastic increase in their d-spacing by a factor of ~4. Temperature, time, and pH influence the swollen interfacial lipid linings. From our study, we conclude that electrostatic interactions introduced by the adsorbed PAH are the main cause for the drastic swelling of the lipid coatings. The DMPC membrane stacks do not detach from their solid support at T > Tm. Steric interactions, also introduced by the PAH molecules, are held responsible for the stabilizing effect. We believe that this novel system offers great potential for fundamental studies of biomembrane properties, keeping the membrane’s natural fluidity and freedom, decoupled from a solid support at physiological conditions. PMID:26703746

  18. Lipid membrane-mediated attraction between curvature inducing objects

    PubMed Central

    van der Wel, Casper; Vahid, Afshin; Šarić, Anđela; Idema, Timon; Heinrich, Doris; Kraft, Daniela J.

    2016-01-01

    The interplay of membrane proteins is vital for many biological processes, such as cellular transport, cell division, and signal transduction between nerve cells. Theoretical considerations have led to the idea that the membrane itself mediates protein self-organization in these processes through minimization of membrane curvature energy. Here, we present a combined experimental and numerical study in which we quantify these interactions directly for the first time. In our experimental model system we control the deformation of a lipid membrane by adhering colloidal particles. Using confocal microscopy, we establish that these membrane deformations cause an attractive interaction force leading to reversible binding. The attraction extends over 2.5 times the particle diameter and has a strength of three times the thermal energy (−3.3 kBT). Coarse-grained Monte-Carlo simulations of the system are in excellent agreement with the experimental results and prove that the measured interaction is independent of length scale. Our combined experimental and numerical results reveal membrane curvature as a common physical origin for interactions between any membrane-deforming objects, from nanometre-sized proteins to micrometre-sized particles. PMID:27618764

  19. Lipid membrane-mediated attraction between curvature inducing objects

    NASA Astrophysics Data System (ADS)

    van der Wel, Casper; Vahid, Afshin; Šarić, Anđela; Idema, Timon; Heinrich, Doris; Kraft, Daniela J.

    2016-09-01

    The interplay of membrane proteins is vital for many biological processes, such as cellular transport, cell division, and signal transduction between nerve cells. Theoretical considerations have led to the idea that the membrane itself mediates protein self-organization in these processes through minimization of membrane curvature energy. Here, we present a combined experimental and numerical study in which we quantify these interactions directly for the first time. In our experimental model system we control the deformation of a lipid membrane by adhering colloidal particles. Using confocal microscopy, we establish that these membrane deformations cause an attractive interaction force leading to reversible binding. The attraction extends over 2.5 times the particle diameter and has a strength of three times the thermal energy (‑3.3 kBT). Coarse-grained Monte-Carlo simulations of the system are in excellent agreement with the experimental results and prove that the measured interaction is independent of length scale. Our combined experimental and numerical results reveal membrane curvature as a common physical origin for interactions between any membrane-deforming objects, from nanometre-sized proteins to micrometre-sized particles.

  20. On the Importance of Hydrodynamic Interactions in Lipid Membrane Formation

    PubMed Central

    Ando, Tadashi; Skolnick, Jeffrey

    2013-01-01

    Hydrodynamic interactions (HI) give rise to collective motions between molecules, which are known to be important in the dynamics of random coil polymers and colloids. However, their role in the biological self-assembly of many molecule systems has not been investigated. Here, using Brownian dynamics simulations, we evaluate the importance of HI on the kinetics of self-assembly of lipid membranes. One-thousand coarse-grained lipid molecules in periodic simulation boxes were allowed to assemble into stable bilayers in the presence and absence of intermolecular HI. Hydrodynamic interactions reduce the monomer-monomer association rate by 50%. In contrast, the rate of association of lipid clusters is much faster in the presence of intermolecular HI. In fact, with intermolecular HI, the membrane self-assembly rate is 3–10 times faster than that without intermolecular HI. We introduce an analytical model to describe the size dependence of the diffusive encounter rate of particle clusters, which can qualitatively explain our simulation results for the early stage of the membrane self-assembly process. These results clearly suggest that HI greatly affects the kinetics of self-assembly and that simulations without HI will significantly underestimate the kinetic parameters of such processes. PMID:23332062

  1. Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide.

    PubMed

    Alvares, Dayane S; Neto, João Ruggiero; Ambroggio, Ernesto E

    2017-03-05

    Polybia-MP1 (IDWKKLLDAAKQIL-NH2) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1.

  2. Lipid-protein interactions in Escherichia coli membranes over-expressing the sugar-H(+) symporter, GalP EPR of spin-labelled lipids.

    PubMed

    Hubert, Anne; Henderson, Peter J F; Marsh, Derek

    2003-04-01

    The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.

  3. Alteration of macrophage membrane lipids following processing of bacterial peptidoglycan

    SciTech Connect

    Polanski, M.; Gray, G.R.

    1986-03-01

    As part of the continuing investigation into the role played by macrophages in antigen presentation and bacterial adjuvant activation, the authors have examined the metabolites produced by macrophages after encounter with peptidoglycan. Peptidoglycan was chosen because it contains N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide), a known adjuvant whose primary target cell is the macrophage. In previous work, the authors established that a series of muramyl dipeptide-like glycopeptides was released into the medium following phagocytosis of peptidoglycan by a macrophage cell line. Here the authors report on the finding that, additionally, a membrane lipid has been covalently altered by the addition of a peptidoglycan fragment. Bacillus subtilis cell walls which had been radiolabeled in their muramic acid, glucosamine and alanine residues, were incubated with the murine macrophage cell line RAW264. Using standard lipid extraction procedures, a lipid was isolated and found to contain equal molar ratios of alanine, glutamic acid and diaminopimelic acid. Since lipidated peptidoglycan peptides have been shown to be immunoactivators, the isolated lipid derivative may serve as a signal for interactions with other lymphocytes.

  4. Lipid Bilayer-Bound Conformation of an Integral Membrane Beta Barrel Protein by Multidimensional MAS NMR

    PubMed Central

    Eddy, Matthew T.; Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay; Wagner, Gerhard; Pintacuda, Guido; Emsley, Lyndon; Griffin, Robert G.

    2015-01-01

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms 2-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13C line width and less than 0.5 ppm 15N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  5. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR.

    PubMed

    Eddy, Matthew T; Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay; Wagner, Gerhard; Pintacuda, Guido; Emsley, Lyndon; Griffin, Robert G

    2015-04-01

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  6. Anomalous surface diffusion of protons on lipid membranes.

    PubMed

    Wolf, Maarten G; Grubmüller, Helmut; Groenhof, Gerrit

    2014-07-01

    The cellular energy machinery depends on the presence and properties of protons at or in the vicinity of lipid membranes. To asses the energetics and mobility of a proton near a membrane, we simulated an excess proton near a solvated DMPC bilayer at 323 K, using a recently developed method to include the Grotthuss proton shuttling mechanism in classical molecular dynamics simulations. We obtained a proton surface affinity of -13.0 ± 0.5 kJ mol(-1). The proton interacted strongly with both lipid headgroup and linker carbonyl oxygens. Furthermore, the surface diffusion of the proton was anomalous, with a subdiffusive regime over the first few nanoseconds, followed by a superdiffusive regime. The time- and distance dependence of the proton surface diffusion coefficient within these regimes may also resolve discrepancies between previously reported diffusion coefficients. Our simulations show that the proton anomalous surface diffusion originates from restricted diffusion in two different surface-bound states, interrupted by the occasional bulk-mediated long-range surface diffusion. Although only a DMPC membrane was considered in this work, we speculate that the restrictive character of the on-surface diffusion is highly sensitive to the specific membrane conditions, which can alter the relative contributions of the surface and bulk pathways to the overall diffusion process. Finally, we discuss the implications of our findings for the energy machinery.

  7. A Critical Reassessment of Penetratin Translocation Across Lipid Membranes

    PubMed Central

    Bárány-Wallje, Elsa; Keller, Sandro; Serowy, Steffen; Geibel, Sebastian; Pohl, Peter; Bienert, Michael; Dathe, Margitta

    2005-01-01

    Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10−13 m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides. PMID:16040762

  8. Interaction measurement of particles bound to a lipid membrane

    NASA Astrophysics Data System (ADS)

    Sarfati, Raphael; Dufresne, Eric

    2015-03-01

    The local shape and dynamics of the plasma membrane play important roles in many cellular processes. Local membrane deformations are often mediated by the adsorption of proteins (notably from the BAR family), and their subsequent self-assembly. The emerging hypothesis is that self-assembly arises from long-range interactions of individual proteins through the membrane's deformation field. We study these interactions in a model system of micron-sized colloidal particles adsorbed onto a lipid bilayer. We use fluorescent microscopy, optical tweezers and particle tracking to measure dissipative and conservative forces as a function of the separation between the particles. We find that particles are driven together with forces of order 100 fN and remain bound in a potential well with a stiffness of order 100 fN/micron.

  9. Elasto-plasticity in wrinkled polymerized lipid membranes

    NASA Astrophysics Data System (ADS)

    Chaieb, Sahraoui

    2014-01-01

    Biomembranes shown to behave like elastic sheets, can also suffer plastic deformations. Neutron scattering experiments on partially polymerised wrinkled membranes revealed that when a critical degree of polymerisation is crossed, the wrinkled membranes do not resume their spherical shapes. Instead they remain wrinkled and rigid while their non-polymerised counterparts resume their spherical floppy shapes. The yield stress of these membranes, measured for the first time via the fractal dimension, is intimately related to the degree of polymerisation probably through a 2D disorder that quenches the lateral diffusion of the lipid molecules. This work might shed light on the physical reason behind the irreversible deformation of echinocytes, acanthocytes and malaria infected red blood cells.

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

    PubMed

    Syed, Aleem; Smith, Emily A

    2017-03-15

    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. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 10 is June 12, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  11. Independent adsorption of monovalent cations and cationic polymers at PE/PG lipid membranes

    NASA Astrophysics Data System (ADS)

    Khomich, Daria A.; Nesterenko, Alexey M.; Kostritskii, Andrei Yu; Kondinskaia, Diana A.; Ermakov, Yuri A.; Gurtovenko, Andrey A.

    2017-01-01

    Synthetic cationic polymers constitute a wide class of polymeric biocides. Commonly their antimicrobial effect is associated to their interaction with bacterial membranes. In the present study we analyze the interaction of various cationic polymers with model bacterial membranes comprised of a mixture of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). We describe a polymer-membrane interaction as a process of modification of the surface charge. It is well known that small monovalent inorganic cations (Na+, K+) cannot overcharge the surface of a bilayer containing anionic lipids. In contrast, polycations are able to overcharge anionic membranes and demonstrate a very large input to the electric field distribution at the membrane-water interface. We aimed here to study the electrostatic effects associated with the interaction of polycations of different types with a model lipid membrane whose composition closely resembles that of bacterial membranes (PE:PG = 1:4). Four different cationic polymers (polyvinylamine, polyallylamine, poly-L-lysine and polyethylenimine) were adsorbed at a model PE/PG bilayer in MD simulations. Adsorption of sodium cations was inspected separately for PE/PG bilayers of different composition and cation’s binding parameters were determined. From computational experiments and consequent theoretical analysis we concluded that sodium adsorption at anionic binding sites does not depend on the presence of polycations. Therefore, we hypothesize that antimicrobial activity of the studied cationic polymers should depend on the ionic composition of the medium.

  12. Genetic Analysis of Arabidopsis Mutants Impaired in Plastid Lipid Import Reveals a Role of Membrane Lipids in Chloroplast Division

    SciTech Connect

    Fan, J.; Xu, C.

    2011-03-01

    The biogenesis of photosynthetic membranes in plants relies largely on lipid import from the endoplasmic reticulum (ER) and this lipid transport process is mediated by TGD proteins in Arabidopsis. Such a dependency of chloroplast biogenesis on ER-to-plastid lipid transport was recently exemplified by analyzing double mutants between tgd1-1 or tgd4-3 and fad6 mutants. The fad6 mutants are defective in the desaturation of membrane lipids in chloroplasts and therefore dependent on import of polyunsaturated lipid precursors from the ER for constructing a competent thylakoid membrane system. In support of a critical role of TGD proteins in ER-to-plastid lipid trafficking, we showed that the introduction of the tgd mutations into fad6 mutant backgrounds led to drastic reductions in relative amounts of thylakoid lipids. Moreover, the tgd1-1 fad6 and tgd4-3 fad6 double mutants were deficient in polyunsaturated fatty acids in chloroplast membrane lipids, and severely compromised in the biogenesis of photosynthetic membrane systems. Here we report that these double mutants are severely impaired in chloroplast division. The possible role of membrane lipids in chloroplast division is discussed.

  13. Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

    PubMed Central

    Owen, Dylan M.; Gaus, Katharina

    2013-01-01

    The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes. PMID:24376453

  14. Kinetics of enzymatic reactions in lipid membranes containing domains.

    PubMed

    Zhdanov, Vladimir P; Höök, Fredrik

    2015-03-06

    An appreciable part of enzymes operating in vivo is associated with lipid membranes. The function of such enzymes can be influenced by the presence of domains containing proteins and/or composed of different lipids. The corresponding experimental model-system studies can be performed under well controlled conditions, e.g., on a planar supported lipid bilayer or surface-immobilized vesicles. To clarify what may happen in such systems, we propose general kinetic equations describing the enzyme-catalyzed substrate conversion occurring via the Michaelis-Menten (MM) mechanism on a membrane with domains which do not directly participate in reaction. For two generic situations when a relatively slow reaction takes place primarily in or outside domains, we take substrate saturation and lateral substrate-substrate interactions at domains into account and scrutinize the dependence of the reaction rate on the average substrate coverage. With increasing coverage, depending on the details, the reaction rate reaches saturation via an inflection point or monotonously as in the conventional MM case. In addition, we show analytically the types of reaction kinetics occurring primarily at domain boundaries. In the physically interesting situation when the domain growth is fast on the reaction time scale, the latter kinetics are far from conventional. The opposite situation when the reaction is fast and controlled by diffusion has been studied by using the Monte Carlo technique. The corresponding results indicate that the dependence of the reaction kinetics on the domain size may be weak.

  15. Redistribution of Cholesterol in Model Lipid Membranes in Response to the Membrane-Active Peptide Alamethicin

    NASA Astrophysics Data System (ADS)

    Heller, William; Qian, Shuo

    2013-03-01

    The cellular membrane is a heterogeneous, dynamic mixture of molecules and macromolecules that self-assemble into a tightly-regulated functional unit that provides a semipermeable barrier between the cell and its environment. Among the many compositional differences between mammalian and bacterial cell membranes that impact its physical properties, one key difference is cholesterol content, which is more prevalent in mammals. Cholesterol is an amphiphile that associates with membranes and serves to maintain its fluidity and permeability. Membrane-active peptides, such as the alpha-helical peptide alamethicin, interact with membranes in a concentration- and composition-dependent manner to form transmembrane pores that are responsible for the lytic action of the peptide. Through the use of small-angle neutron scattering and deuterium labeling, it was possible to observe a redistribution of the lipid and cholesterol in unilamellar vesicles in response to the presence of alamethicin at a peptide-to-lipid ratio of 1/200. The results demonstrate that the membrane remodeling powers of alamethicin reach beyond the membrane thinning effect to altering the localization of specific components in the bilayer, complementing the accepted two-state mechanism of pore formation. Research was supported by U. S. DOE-OBER (CSMB; FWP ERKP291) and the U. S. DOE-BES Scientific User Facilities Division (ORNL's SNS and HFIR).

  16. An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

    PubMed

    Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

    2014-01-16

    Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption.

  17. Membrane on a chip: a functional tethered lipid bilayer membrane on silicon oxide surfaces.

    PubMed

    Atanasov, Vladimir; Knorr, Nikolaus; Duran, Randolph S; Ingebrandt, Sven; Offenhäusser, Andreas; Knoll, Wolfgang; Köper, Ingo

    2005-09-01

    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.

  18. Membrane lipid peroxidation by UV-A: Mechanism and implications

    SciTech Connect

    Bose, B.; Agarwal, S.; Chatterjee, S.N. )

    1990-10-01

    UV-A produced a dose-dependent linear increase of lipid peroxidation in liposomal membrane, as detected by the assay of (i) conjugated dienes, (ii) lipid hydroperoxides, (iii) malondialdehydes (MDA), and (iv) the fluorescent adducts formed by the reaction of MDA with glycine and also a linear dose-dependent increase of ({sup 14}C)glucose efflux from the liposomes. UV-A-induced MDA production could not be inhibited by any significant degree by sodium formate, dimethyl sulfoxide, EDTA, or superoxide dismutase but was very significantly inhibited by butylated hydroxytoluene, alpha-tocopherol, sodium azide, L-histidine, dimethylfuran, and beta-carotene. MDA formation increased with an increase in the D{sub 2}O content in water, leading to a maximal amount of nearly 50% enhancement of lipid peroxidation in 100% D{sub 2}O vis-a-vis water used as dispersion medium. The experimental findings indicate the involvement of singlet oxygen as the initiator of the UV-A-induced lipid peroxidation.

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

    PubMed

    Alekseev, S I; Ziskin, M C

    1995-01-01

    The effects of millimeter microwaves in the frequency range of 54-76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB-). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% +/- 0.5%. At the same time, membrane current induced by TPhB- transport increased by 5% +/- 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% +/- 0.4%). No "resonance-like" effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB- transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 degrees C.

  20. Solubility and Permeation of Hydrogen Sulfide in Lipid Membranes

    PubMed Central

    Cuevasanta, Ernesto; Denicola, Ana; Alvarez, Beatriz; Möller, Matías N.

    2012-01-01

    Hydrogen sulfide (H2S) is mainly known for its toxicity but has recently been shown to be produced endogenously in mammalian tissues and to be associated with physiological regulatory functions. To better understand the role of biomembranes in modulating its biological distribution and effects; we measured the partition coefficient of H2S in models of biological membranes. The partition coefficients were found to be 2.1±0.2, 1.9±0.5 and 2.0±0.6 in n-octanol, hexane and dilauroylphosphatidylcholine liposome membranes relative to water, respectively (25°C). This two-fold higher concentration of H2S in the membrane translates into a rapid membrane permeability, Pm = 3 cm s−1. We used a mathematical model in three dimensions to gain insight into the diffusion of total sulfide in tissues. This model shows that the sphere of action of sulfide produced by a single cell expands to involve more than 200 neighboring cells, and that the resistance imposed by lipid membranes has a significant effect on the diffusional spread of sulfide at pH 7.4, increasing local concentrations. These results support the role of hydrogen sulfide as a paracrine signaling molecule and reveal advantageous pharmacokinetic properties for its therapeutic applications. PMID:22509322

  1. Biological Membranes in Extreme Conditions: Simulations of Anionic Archaeal Tetraether Lipid Membranes

    PubMed Central

    Pineda De Castro, Luis Felipe; Dopson, Mark

    2016-01-01

    In contrast to the majority of organisms that have cells bound by di-ester phospholipids, archaeal membranes consist of di- and tetraether phospholipids. Originating from organisms that withstand harsh conditions (e.g., low pH and a wide range of temperatures) such membranes have physical properties that make them attractive materials for biological research and biotechnological applications. We developed force-field parameters based on the widely used Generalized Amber Force Field (GAFF) to enable the study of anionic tetraether membranes of the model archaean Sulfolobus acidocaldarius by computer simulations. The simulations reveal that the physical properties of these unique membranes depend on the number of cyclopentane rings included in each lipid unit, and on the size of cations that are used to ensure charge neutrality. This suggests that the biophysical properties of Sulfolobus acidocaldarius cells depend not only on the compositions of their membranes but also on the media in which they grow. PMID:27167213

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

    SciTech Connect

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

    1988-02-17

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

  3. Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes.

    PubMed

    Tan, Anmin; Ziegler, André; Steinbauer, Bernhard; Seelig, Joachim

    2002-09-01

    The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at

  4. Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

    PubMed Central

    Konshina, Anastasia G.; Boldyrev, Ivan A.; Utkin, Yuri N.; Omel'kov, Anton V.; Efremov, Roman G.

    2011-01-01

    The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of

  5. Electroporation of archaeal lipid membranes using MD simulations.

    PubMed

    Polak, Andraž; Tarek, Mounir; Tomšič, Matija; Valant, Janez; Ulrih, Nataša Poklar; Jamnik, Andrej; Kramar, Peter; Miklavčič, Damijan

    2014-12-01

    Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91 mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343 K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2 V to 4.3 V at 323 K, and to 5.2 V at 298 K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5 V at 323 K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50 mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8 V and 4.1 V at respectively 323 and 298 K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.

  6. Direct observation of brownian motion of lipids in a membrane.

    PubMed Central

    Lee, G M; Ishihara, A; Jacobson, K A

    1991-01-01

    Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 10(-8) cm2/sec, and paucivalent probes had an average D of 0.73 x 10(-8) cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 x 10(-8) cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 x 10(-8) cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results. Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes. Images PMID:1712486

  7. Clinorotation Effect on Coupling Level and Lipid Composition of Barley Thylakoid Membranes

    NASA Astrophysics Data System (ADS)

    Mykhaylenko, N.; Podorvano, V.; Zolotareva, O.

    Microgravity can induce structural perturbation of plant photosynthetic apparatus. It was shown that space flight conditions caused both pigment content and chloroplast ultrastructure changes in a number of various plant species. The transformations of photosynthetic membrane lipid composition were observed in wheat plants under microgravity as well as in chloroplasts of pea under clinorotation. The photosynthetic apparatus is located in thylakoid membranes of chloroplast and provides plant cell by macroerg compounds (ATP and NADPH) necessary for inorganic carbon fixation and metabolism. ATP is formed in the process of photophosphorylation, the rate of which is determined by a coupling level of thylakoid membranes. The aim of the work was to study the coupling level and lipid composition of thylakoid membranes isolated from barley plants grown under clinorotation. Plants of barley (Hordeum vulgare L.) were grown for 7 days at 22-24°C, at low illumination (143 μ mol m-2 s-1) with a light period of 16 h, on a horizontal clinostat (2 rpm) and in vertical control. Photochemical activity of isolated chloroplasts (class II) was estimated by the following reactions: cyclic and non-cyclic photophosphorylation, coupled and uncoupled electron transfer from water to K3Fe(CN)6. Total lipids were extracted from isolated chloroplasts and individual lipid classes were separated by thin-layer chromatography. Phospholipids were determined in the form of inorganic phosphate after mineralization with perchloric acid. Glycolipids were assayed by monosaccharide content after acidic hydrolysis. Gas chromatography was applied to analyse the fatty acid composition of membrane glycerolipids. The rates of both cyclic and non-cyclic photophosphorylation in chloroplasts isolated from clinorotated plants were lower than those in control samples. At the same time the rate of electron transfer in thylakoid membranes from clinorotated plants was higher. In the presence of protonophoric channel

  8. Ionization behavior of amino lipids for siRNA delivery: determination of ionization constants, SAR, and the impact of lipid pKa on cationic lipid-biomembrane interactions.

    PubMed

    Zhang, Jingtao; Fan, Haihong; Levorse, Dorothy A; Crocker, Louis S

    2011-03-01

    Ionizable amino lipids are being pursued as an important class of materials for delivering small interfering RNA (siRNA) therapeutics, and research is being conducted to elucidate the structure-activity relationships (SAR) of these lipids. The pK(a) of cationic lipid headgroups is one of the critical physiochemical properties of interest due to the strong impact of lipid ionization on the assembly and performance of these lipids. This research focused on developing approaches that permit the rapid determination of the relevant pK(a) of the ionizable amino lipids. Two distinct approaches were investigated: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye to cationic liposomes formulated from amino lipids. Using the approaches developed here, the pK(a) values of cationic lipids with distinct headgroups were measured and found to be significantly lower than calculated values. It was also found that lipid-lipid interaction has a strong impact on the pK(a) values of lipids. Lysis of model biomembranes by cationic lipids was used to evaluate the impact of lipid pK(a) on the interaction between cationic lipids and cell membranes. It was found that cationic lipid-biomembrane interaction depends strongly on lipid pK(a) and solution pH, and this interaction is much stronger when amino lipids are highly charged. The presence of an optimal pK(a) range of ionizable amino lipids for siRNA delivery was suggested based on these results. The pK(a) methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pK(a) on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.

  9. Lipid-Modulated Sequence-Specific Association of Glycophorin A in Membranes

    PubMed Central

    Janosi, Lorant; Prakash, Anupam; Doxastakis, Manolis

    2010-01-01

    Abstract Protein association in lipid membranes is a complex process with thermodynamics directed by a multitude of different factors. Amino-acid sequence is a molecular parameter that affects dimerization as shown by limited directed mutations along the transmembrane domains. Membrane-mediated interactions are also important although details of such contributions remain largely unclear. In this study, we probe directly the free energy of association of Glycophorin A by means of extensive parallel Monte Carlo simulations with recently developed methods and a model that accounts for sequence-specificity while representing lipid membranes faithfully. We find that lipid-induced interactions are significant both at short and intermediate separations. The ability of molecules to tilt in a specific hydrophobic environment extends their accessible interfaces, leading to intermittent contacts during protein recognition. The dimer with the lowest free energy is largely determined by the favorable lipid-induced attractive interactions at the closest distance. Finally, the coarse-grained model employed herein, together with the extensive sampling performed, provides estimates of the free energy of association that are in excellent agreement with existing data. PMID:20655857

  10. Length and sequence dependence in the association of Huntingtin protein with lipid membranes

    NASA Astrophysics Data System (ADS)

    Jawahery, Sudi; Nagarajan, Anu; Matysiak, Silvina

    2013-03-01

    There is a fundamental gap in our understanding of how aggregates of mutant Huntingtin protein (htt) with overextended polyglutamine (polyQ) sequences gain the toxic properties that cause Huntington's disease (HD). Experimental studies have shown that the most important step associated with toxicity is the binding of mutant htt aggregates to lipid membranes. Studies have also shown that flanking amino acid sequences around the polyQ sequence directly affect interactions with the lipid bilayer, and that polyQ sequences of greater than 35 glutamine repeats in htt are a characteristic of HD. The key steps that determine how flanking sequences and polyQ length affect the structure of lipid bilayers remain unknown. In this study, we use atomistic molecular dynamics simulations to study the interactions between lipid membranes of varying compositions and polyQ peptides of varying lengths and flanking sequences. We find that overextended polyQ interactions do cause deformation in model membranes, and that the flanking sequences do play a role in intensifying this deformation by altering the shape of the affected regions.

  11. Influences of the Structure of Lipids on Thermal Stability of Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Hai, Nan-Nan; Zhou, Xin; Li, Ming

    2015-08-01

    The binding free energy (BFE) of lipid to lipid bilayer is a critical factor to determine the thermal or mechanical stability of the bilayer. Although the molecular structure of lipids has significant impacts on BFE of the lipid, there lacks a systematic study on this issue. In this paper we use coarse-grained molecular dynamics simulation to investigate this problem for several typical phospholipids. We find that both the tail length and tail unsaturation can significantly affect the BFE of lipids but in opposite way, namely, BFE decreases linearly with increasing length, but increases linearly with addition of unsaturated bonds. Inspired by the specific structure of cholesterol which is a crucial component of biomembrane, we also find that introduction of carbo-ring-like structures to the lipid tail or to the bilayer may greatly enhance the stability of the bilayer. Our simulation also shows that temperature can influence the bilayer stability and this effect can be significant when the bilayer undergoes phase transition. These results may be helpful to the design of liposome or other self-assembled lipid systems. Support by the National Natural Science Foundation of China under Grant Nos. 91027046 and 11105218.

  12. Cryoprotection of Lipid Membranes for High-Resolution Solid-State NMR Studies of Membrane Peptides and Proteins at Low Temperature

    PubMed Central

    Lee, Myungwoon; Hong, Mei

    2014-01-01

    Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. 13C and 1H MAS spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. 13C-1H dipolar couplings and 31P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work – DMSO, PEG, and DMF - should be useful for low

  13. Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature.

    PubMed

    Lee, Myungwoon; Hong, Mei

    2014-08-01

    Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. 13C and 1H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. 13C-1H dipolar couplings and 31P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work-DMSO, PEG, and DMF-should be useful for low

  14. 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.

  15. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed

    Colarusso, Pina; Spring, Kenneth R

    2002-02-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane.

  16. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed Central

    Colarusso, Pina; Spring, Kenneth R

    2002-01-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane. PMID:11806917

  17. Detergent interaction with tethered bilayer lipid membranes for protein reconstitution

    NASA Astrophysics Data System (ADS)

    Broccio, Matteo; Zan Goh, Haw; Loesche, Mathias

    2009-03-01

    Tethered bilayer lipid membranes (tBLMs) are self-assembled biomimetic structures in which the membrane is separated from a solid substrate by a nm-thick hydrated submembrane space. These model systems are being used in binding studies of peripheral proteins and exotoxins. Here we aim at their application for the reconstitution of water-insoluble integral membrane proteins. As an alternative to fusion of preformed proteoliposomes we study the direct reconstitution of such proteins for applications in biosensing and pharmaceutical screening. For reconstitution, highly insulating tBLMs (R˜10^5-10^6 φ) were temporarily incubated with a detergent to screen for conditions that keep the detergent-saturated membranestable and ready to incorporate detergent-solubilized proteins. We assess the electrical characteristics, i.e. specific resistance and capacitance, by means of electrochemical impedance spectroscopy (EIS) under timed incubation with decylmaltoside and dodecylmaltoside detergents in a regime around their critical micelle concentration, 1.8 mM and 0.17 mM respectively and demonstrate the restoration of the tBLM upon detergent removal. Thereby a range of concentration and incubation times was identified, that represents optimal conditions for the subsequent membrane protein reconstitution.

  18. Effects of seaweed sterols fucosterol and desmosterol on lipid membranes.

    PubMed

    Mouritsen, Ole G; Bagatolli, Luis A; Duelund, Lars; Garvik, Olav; Ipsen, John H; Simonsen, Adam Cohen

    2017-03-30

    Higher sterols are universally present in large amounts (20-30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.

  19. Statistical thermodynamic analysis of peptide and protein insertion into lipid membranes.

    PubMed Central

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

    1996-01-01

    A statistical thermodynamic approach is used to analyze the various contributions to the free energy change associated with the insertion of proteins and protein fragments into lipid bilayers. The partition coefficient that determines the equilibrium distribution of proteins between the membrane and the solution is expressed as the ratio between the partition functions of the protein in the two phases. It is shown that when all of the relevant degrees of freedom (i.e., those that change their character upon insertion into the membrane) can be treated classically, the partition coefficient is fully determined by the ratio of the configurational integrals and thus does not involve any mass-dependent factors, a conclusion that is also valid for related processes such as protein adsorption on a membrane surface or substrate binding to proteins. The partition coefficient, and hence the transfer free energy, depend only on the potential energy of the protein in the membrane. Expressing this potential as a sum of a "static" term, corresponding to the equilibrium (minimal free energy) configuration of the protein in the membrane, and a "dynamical" term representing fluctuations around the equilibrium configuration, we show that the static term contains the "solvation" and "lipid perturbation" contributions to the transfer free energy. The dynamical term is responsible for the "immobilization" free energy, reflecting the loss of translational and rotational entropy of the protein upon incorporation into the membrane. Based on a recent molecular theory of lipid-protein interactions, the lipid perturbation and immobilization contributions are then expressed in terms of the elastic deformation free energy resulting from the perturbation of the lipid environment by the foreign (protein) inclusion. The model is formulated for cylindrically shaped proteins, and numerical estimates are given for the insertion of an alpha-helical peptide into a lipid bilayer. The immobilization

  20. Probing the importance of lipid diversity in cell membranes via molecular simulation.

    PubMed

    Khakbaz, Pouyan; Klauda, Jeffery B

    2015-11-01

    Lipid membranes in prokaryotes and eukaryotes have a wide array of lipids that are necessary for proper membrane structure and function. In this paper, an introduction to lipid diversity in biology and a mini-review on how molecular simulations have been used to model biological membranes (primarily limited to one to three lipid types in most simulation-based models) is provided, which motivates the use of all-atom molecular dynamics (MD) simulations to study the effect of lipid diversity on properties of realistic membrane models of prokaryotes and eukaryotes. As an example, cytoplasmic membrane models of Escherichia coli were developed at different stages of the colony growth cycle (early-log, mid-log, stationary and overnight). The main difference between lipid compositions at each stage was the concentration of a cyclopropane-containing moiety on the sn-2 lipid acyl chain (cyC17:0). Triplicate MD simulations for each stage were run for 300 ns to study the influence of lipid diversity on the surface area per lipid, area compressibility modulus, deuterium order parameters, and electron density profiles. The overnight stage (also known as the death stage) had the highest average surface area per lipid, highest rigidity, and lowest bilayer thickness compare to other stages of E. coli cytoplasmic membrane. Although bilayer thickness did depend on the growth stage, the changes between these were small suggesting that the hydrophobic core of transmembrane proteins fit well with the membrane in all growth stages. Although it is still common practise in MD simulations of membrane proteins to use simple one- or two-component membranes, it can be important to use diverse lipid model membranes when membrane protein structure and function are influenced by changes in lipid membrane composition.

  1. Proving lipid rafts exist: membrane domains in the prokaryote Borrelia burgdorferi have the same properties as eukaryotic lipid rafts.

    PubMed

    LaRocca, Timothy J; Pathak, Priyadarshini; Chiantia, Salvatore; Toledo, Alvaro; Silvius, John R; Benach, Jorge L; London, Erwin

    2013-01-01

    Lipid rafts in eukaryotic cells are sphingolipid and cholesterol-rich, ordered membrane regions that have been postulated to play roles in many membrane functions, including infection. We previously demonstrated the existence of cholesterol-lipid-rich domains in membranes of the prokaryote, B. burgdorferi, the causative agent of Lyme disease [LaRocca et al. (2010) Cell Host & Microbe 8, 331-342]. Here, we show that these prokaryote membrane domains have the hallmarks of eukaryotic lipid rafts, despite lacking sphingolipids. Substitution experiments replacing cholesterol lipids with a set of sterols, ranging from strongly raft-promoting to raft-inhibiting when mixed with eukaryotic sphingolipids, showed that sterols that can support ordered domain formation are both necessary and sufficient for formation of B. burgdorferi membrane domains that can be detected by transmission electron microscopy or in living organisms by Förster resonance energy transfer (FRET). Raft-supporting sterols were also necessary and sufficient for formation of high amounts of detergent resistant membranes from B. burgdorferi. Furthermore, having saturated acyl chains was required for a biotinylated lipid to associate with the cholesterol-lipid-rich domains in B. burgdorferi, another characteristic identical to that of eukaryotic lipid rafts. Sterols supporting ordered domain formation were also necessary and sufficient to maintain B. burgdorferi membrane integrity, and thus critical to the life of the organism. These findings provide compelling evidence for the existence of lipid rafts and show that the same principles of lipid raft formation apply to prokaryotes and eukaryotes despite marked differences in their lipid compositions.

  2. 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.

  3. Structural basis for the transcriptional regulation of membrane lipid homeostasis

    SciTech Connect

    Miller, Darcie J.; Zhang, Yong-Mei; Subramanian, Chitra; Rock, Charles O.; White, Stephen W.

    2010-11-09

    DesT is a transcriptional repressor that regulates the genes that control the unsaturated:saturated fatty acid ratio available for membrane lipid synthesis. DesT bound to unsaturated acyl-CoA has a high affinity for its cognate palindromic DNA-binding site, whereas DesT bound to saturated acyl-CoA does not bind this site. Structural analyses of the DesT-oleoyl-CoA-DNA and DesT-palmitoyl-CoA complexes reveal that acyl chain shape directly influences the packing of hydrophobic core residues within the DesT ligand-binding domain. These changes are propagated to the paired DNA-binding domains via conformational changes to modulate DNA binding. These structural interpretations are supported by the in vitro and in vivo characterization of site-directed mutants. The regulation of DesT by the unsaturated:saturated ratio of acyl chains rather than the concentration of a single ligand is a paradigm for understanding transcriptional regulation of membrane lipid homeostasis.

  4. Interaction of aldehydes derived from lipid peroxidation and membrane proteins

    PubMed Central

    Pizzimenti, Stefania; Ciamporcero, Eric; Daga, Martina; Pettazzoni, Piergiorgio; Arcaro, Alessia; Cetrangolo, Gianpaolo; Minelli, Rosalba; Dianzani, Chiara; Lepore, Alessio; Gentile, Fabrizio; Barrera, Giuseppina

    2013-01-01

    A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA) and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE) is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation, and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions. PMID:24027536

  5. Tight binding of NAP-22 with acidic membrane lipids.

    PubMed

    Maekawa, Shohei; Kobayashi, Yuumi; Morita, Mitsuhiro; Suzaki, Toshinobu

    2015-07-23

    Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. Insolubility of the outer leaflet of DRM to the non-ionic detergent is ascribed to the tight association of cholesterol and sphingolipid. Since, poor localization of sphingolipid is observed in the inner leaflet, the physicochemical background of the insolubility of the inner leaflet is hence still an enigma. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM of the neuronal cell membrane. A previous study showed the presence of several lipids in a NAP-22 fraction after the process of extraction and column chromatography. In this study, the effect of lipid extraction on NAP-22 was studied through native-gel electrophoresis, ultracentrifugation, and electron microscopic observation. The mobility of NAP-22 in native-PAGE was shifted from low to high after delipidation. Delipidated NAP-22 bound phosphatidylserine (PS), phosphatidylinosotol, and ganglioside. Some part of the mixture of PS and NAP-22 was recovered in the insoluble fraction after Triton X-100 treatment and the addition of cholesterol enhanced the amount of NAP-22 in the insoluble fraction.

  6. Vascular endothelial cell membranes differentiate between stretch and shear stress through transitions in their lipid phases.

    PubMed

    Yamamoto, Kimiko; Ando, Joji

    2015-10-01

    Vascular endothelial cells (ECs) respond to the hemodynamic forces stretch and shear stress by altering their morphology, functions, and gene expression. However, how they sense and differentiate between these two forces has remained unknown. Here we report that the plasma membrane itself differentiates between stretch and shear stress by undergoing transitions in its lipid phases. Uniaxial stretching and hypotonic swelling increased the lipid order of human pulmonary artery EC plasma membranes, thereby causing a transition from the liquid-disordered phase to the liquid-ordered phase in some areas, along with a decrease in membrane fluidity. In contrast, shear stress decreased the membrane lipid order and increased membrane fluidity. A similar increase in lipid order occurred when the artificial lipid bilayer membranes of giant unilamellar vesicles were stretched by hypotonic swelling, indicating that this is a physical phenomenon. The cholesterol content of EC plasma membranes significantly increased in response to stretch but clearly decreased in response to shear stress. Blocking these changes in the membrane lipid order by depleting membrane cholesterol with methyl-β-cyclodextrin or by adding cholesterol resulted in a marked inhibition of the EC response specific to stretch and shear stress, i.e., phosphorylation of PDGF receptors and phosphorylation of VEGF receptors, respectively. These findings indicate that EC plasma membranes differently respond to stretch and shear stress by changing their lipid order, fluidity, and cholesterol content in opposite directions and that these changes in membrane physical properties are involved in the mechanotransduction that activates membrane receptors specific to each force.

  7. Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion.

    PubMed Central

    Fahsel, S; Pospiech, E-M; Zein, M; Hazlet, T L; Gratton, E; Winter, Roland

    2002-01-01

    The lateral membrane organization and phase behavior of the binary lipid mixture DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) - DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) without and with incorporated gramicidin D (GD) as a model biomembrane polypeptide was studied by small-angle neutron scattering, Fourier-transform infrared spectroscopy, and by two-photon excitation fluorescence microscopy on giant unilamellar vesicles. The small-angle neutron scattering method allows the detection of concentration fluctuations in the range from 1 to 200 nm. Fluorescence microscopy was used for direct visualization of the lateral lipid organization and domain shapes on a micrometer length scale including information of the lipid phase state. In the fluid-gel coexistence region of the pure binary lipid system, large-scale concentration fluctuations appear. Infrared spectral parameters were used to determine the peptide conformation adopted in the different lipid phases. The data show that the structure of the temperature-dependent lipid phases is significantly altered by the insertion of 2 to 5 mol% GD. At temperatures corresponding to the gel-fluid phase coexistence region the concentration fluctuations drastically decrease, and we observe domains in the giant unilamellar vesicles, which mainly disappear by the incorporation of 2 to 5 mol% GD. Further, the lipid matrix has the ability to modulate the conformation of the inserted polypeptide. The balance between double-helical and helical dimer structures of GD depends on the phospholipid chain length and phase state. A large hydrophobic mismatch, such as in gel phase one-component DSPC bilayers, leads to an increase in population of double-helical structures. Using an effective molecular sorting mechanism, a large hydrophobic mismatch can be avoided in the DMPC-DSPC lipid mixture, which leads to significant changes in the heterogeneous lipid structure and in polypeptide conformation. PMID:12080124

  8. Analysis of Membrane Lipids of Airborne Micro-Organisms

    NASA Technical Reports Server (NTRS)

    MacNaughton, Sarah

    2006-01-01

    A method of characterization of airborne micro-organisms in a given location involves (1) large-volume filtration of air onto glass-fiber filters; (2) accelerated extraction of membrane lipids of the collected micro-organisms by use of pressurized hot liquid; and (3) identification and quantitation of the lipids by use of gas chromatography and mass spectrometry. This method is suitable for use in both outdoor and indoor environments; for example, it can be used to measure airborne microbial contamination in buildings ("sick-building syndrome"). The classical approach to analysis of airborne micro-organisms is based on the growth of cultureable micro-organisms and does not provide an account of viable but noncultureable micro-organisms, which typically amount to more than 90 percent of the micro-organisms present. In contrast, the present method provides an account of all micro-organisms, including cultureable, noncultureable, aerobic, and anaerobic ones. The analysis of lipids according to this method makes it possible to estimate the number of viable airborne micro-organisms present in the sampled air and to obtain a quantitative profile of the general types of micro-organisms present along with some information about their physiological statuses.

  9. Cholesterol as a factor regulating the influence of natural (PAF and lysoPAF) vs synthetic (ED) ether lipids on model lipid membranes.

    PubMed

    Flasiński, Michał; Wydro, Paweł; Hąc-Wydro, Katarzyna; Dynarowicz-Łątka, Patrycja

    2013-11-01

    In this work we have performed a comparative study on the effect of antineoplastic ether lipid-edelfosine (ED), its natural analogs - Platelet Activating Factor (PAF) and its precursor (lyso-PAF), both lacking anticancer properties, on cholesterol/phosphatidylcholine (Chol/PC) monolayers, serving as model membranes. Since all the above ether lipids are membrane active, it can be expected that their effect on membranes may differentiate their biological activity. Our investigations were aimed at studying potential relationship of the effect of ED, PAF and lyso-PAF on model membranes, differing in condensation. We have modified molecular packing of Chol/PC model systems either by increasing the level of sterol in the system or changing the structure of PC, while keeping the same sterol content. Additionally, we have performed a detailed comparison of the miscibility of ED, PAF and lyso-PAF with various membrane lipids. The collected data evidenced that all the investigated ether lipids influence Chol/PC films in the same way; however, in a different magnitude. Moreover, the interactions of ED, PAF and lyso-PAF with model membranes were the strongest at the highest level of sterol in the system. A thorough analysis of the obtained results has proved that the effect of the investigated ether lipids on membranes is not dependent on the condensation of the system, but it is strongly determined by the concentration of cholesterol. Since ED was found to interact with model membranes stronger than PAF and lyso-PAF, we have suggested that this fact may contribute to differences in cytotoxicity of these compounds.

  10. Renaturing Membrane Proteins in the Lipid Cubic Phase, a Nanoporous Membrane Mimetic

    PubMed Central

    Li, Dianfan; Caffrey, Martin

    2014-01-01

    Membrane proteins play vital roles in the life of the cell and are important therapeutic targets. Producing them in large quantities, pure and fully functional is a major challenge. Many promising projects end when intractable aggregates or precipitates form. Here we show how such unfolded aggregates can be solubilized and the solution mixed with lipid to spontaneously self-assemble a bicontinuous cubic mesophase into the bilayer of which the protein, in a confined, chaperonin-like environment, reconstitutes with 100% efficiency. The test protein, diacylglycerol kinase, reconstituted in the bilayer of the mesophase, was then crystallized in situ by the in meso or lipid cubic phase method providing an X-ray structure to a resolution of 2.55 Å. This highly efficient, inexpensive, simple and rapid approach should find application wherever properly folded, membrane reconstituted and functional proteins are required where the starting material is a denatured aggregate. PMID:25055873

  11. Relationship between the Amount of Bitter Substances Adsorbed onto Lipid/Polymer Membrane and the Electric Response of Taste Sensors

    PubMed Central

    Toko, Kiyoshi; Hara, Daichi; Tahara, Yusuke; Yasuura, Masato; Ikezaki, Hidekazu

    2014-01-01

    The bitterness of bitter substances can be measured by the change in the membrane electric potential caused by adsorption (CPA) using a taste sensor (electronic tongue). In this study, we examined the relationship between the CPA value due to an acidic bitter substance and the amount of the bitter substance adsorbed onto lipid/polymer membranes, which contain different lipid contents, used in the taste sensor. We used iso-α-acid which is an acidic bitter substance found in several foods and beverages. The amount of adsorbed iso-α-acid, which was determined by spectroscopy, showed a maximum at the lipid concentration 0.1 wt % of the membrane, and the same phenomenon was observed for the CPA value. At the higher lipid concentration, however, the amount adsorbed decreased and then remained constant, while the CPA value decreased monotonically to zero. This constant adsorption amount was observed when the membrane potential in the reference solution did not change with increasing lipid concentration. The decrease in CPA value in spite of the constant adsorption amount is caused by a decrease in the sensitivity of the membrane as the surface charge density increases. The reason why the peaks appeared in both the CPA value and adsorption amount is based on the contradictory adsorption properties of iso-α-acid. The increasing charged lipid concentration of the membrane causes an increasing electrostatic attractive interaction between iso-α-acid and the membrane, but simultaneously causes a decreasing hydrophobic interaction that results in decreasing adsorption of iso-α-acid, which also has hydrophobic properties, onto the membrane. Estimates of the amount of adsorption suggest that iso-α-acid molecules are adsorbed onto both the surface and interior of the membrane. PMID:25184491

  12. Remodeling of membrane lipids in iron-starved Chlamydomonas.

    PubMed

    Urzica, Eugen I; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M Dudley; Casero, David; Gallaher, Sean D; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S

    2013-10-18

    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

  13. Remodeling of Membrane Lipids in Iron-starved Chlamydomonas*

    PubMed Central

    Urzica, Eugen I.; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M. Dudley; Casero, David; Gallaher, Sean D.; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S.

    2013-01-01

    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24–48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii. PMID:23983122

  14. Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms.

    PubMed Central

    Nanjundan, M; Possmayer, F

    2001-01-01

    Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics. PMID:11535125

  15. Phase segregation of polymerizable lipids to construct filters for separating lipid-membrane-embedded species

    PubMed Central

    Hu, Shu-Kai; Chen, Ya-Ming; Chao, Ling

    2014-01-01

    Supported lipid bilayer (SLB) platforms have been developed to transport and separate membrane-embedded species in the species' native bilayer environment. In this study, we used the phase segregation phenomenon of lipid mixtures containing a polymerizable diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), and a nonpolymerizable phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), to create filter barrier structures in SLBs. Upon exposing the phase segregated samples to UV light, the DiynePC-rich domains could become crosslinked and remain fixed on the surface of the support, while the DOPC-rich regions, where no crosslinking could happen, could be removed later by detergent washing, and thus became the void regions in the filter. During the filter fabrication process, we used the laminar flow configuration in a microfluidic channel to control the spatial locations of the feed region and filter region in the SLB. The flow in a microfluidic channel was also used to apply a strong hydrodynamic shear stress to the SLB to transport the membrane-embedded species from the feed region to the filter region. We varied the DiynePC/DOPC molar ratio from 60/40 to 80/20 to adjust the cutoff size of the filter barriers and used two model membrane-embedded species of different sizes to examine the filtering capability. One of the model species, Texas Red 1,2-dihexa-decanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (Texas Red DHPE), had a single-lipid size, and the other species, cholera toxin subunit B-GM1 complex, had a multilipid size. When the DiynePC/DOPC molar ratio was 60/40, both species had high penetration ratios in the filter region. However, when the ratio was increased to 70/30, only the Texas Red DHPE, which was the smaller of the two model species, could penetrate the filter to a considerable extent. When the ratio was increased to 80/20, neither of the model species could penetrate the filter

  16. Plasma zinc status and membrane lipid composition in genetically diabetic mice (db/db)

    SciTech Connect

    Burke, J.P.; Fenton, M.R.

    1986-03-05

    Sex and age matched diabetic C57BL/Ks-db+/db+ mice (db/db) were sacrificed at eight weeks of age. Plasma samples were collected and zinc levels determined. Livers were excised and mitochondrial and microsomal membranes prepared. Aliquots of membrane fractions were subjected to lipid extraction and cholesterol (Cl), phospholipid (PL) and fatty acid analysis (FA) performed. Plasma zinc levels in db/db mice were elevated 25% compared to m/m controls (148.8+/-8.1 ..mu..g/dl vs. 118.9+/-14.9 ..mu..g/dl). Cholesterol and PL levels remained unchanged in both mitochondrial and microsomal membranes. Analysis of PL composition from db/db mitochondria by two dimensional thin layer chromatography revealed no change in the percentage of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) but a 40% decrease in cardiolipin. Slight increases were observed in the percentage of phosphatidylserine and phosphatidylinositol (PS+PI) in microsomes isolated from db/db mice. Fatty acid analysis of microsomal PC and PE showed a decrease of 28% in the 18:1/18:0 ratio as well as a 21% decrease in the ratio of 20:4/18:2 in db/db animals. Analysis of succinate dehydrogenase (mitochondrial) and glucose-6-phosphatase (microsomal) revealed significant decreases in activity in livers of db/db mice. The altered zinc metabolism as well as the changes in membrane lipid composition suggest that this may be a model to study the role of zinc in membrane structure.

  17. Interaction of poloxamine block copolymers with lipid membranes: Role of copolymer structure and membrane cholesterol content.

    PubMed

    Sandez-Macho, Isabel; Casas, Matilde; Lage, Emilio V; Rial-Hermida, M Isabel; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2015-09-01

    Interactions of X-shaped poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers with cell membranes were investigated recording the π-A isotherms of monolayer systems of dipalmitoylphosphatidylcholine (DPPC):cholesterol 100:0; 80:20 and 60:40 mol ratio and evaluating the capability of the copolymers to trigger haemolysis or to protect from haemolytic agents. Four varieties of poloxamine (Tetronic 904, 908, 1107 and 1307) were chosen in order to cover a wide range of EO and PO units contents and molecular weights, and compared to a variety of poloxamer (Pluronic P85). The π-A isotherms revealed that the greater the content in cholesterol, the stronger the interaction of the block copolymers with the lipids monolayer. The interactions were particularly relevant at low pressures and low lipid proportions, mimicking the conditions of damaged membranes. Relatively hydrophobic copolymers bearing short PEO blocks (e.g., T904 and P85) intercalated among the lipids expanding the surface area (ΔGexc) but not effectively sealing the pores. These varieties showed haemolytic behavior. Oppositely, highly hydrophilic copolymers bearing long PEO blocks (e.g., T908, T1107 and T1307) caused membrane contraction and outer leaflet sealing due to strong interactions of PEO with cholesterol and diamine core with phospholipids. These later varieties were not haemolytic and exerted a certain protective effect against spontaneous haemolysis for both intact erythrocytes and cholesterol-depleted erythrocytes.

  18. Membrane-mimetic films of asymmetric phosphatidylcholine lipid bolaamphiphiles.

    PubMed

    Sun, Xue-Long; Biswas, Nilanjana; Kai, Toshitsugu; Dai, Zhifei; Dluhy, Richard A; Chaikof, Elliot L

    2006-01-31

    Membrane-spanning phospholipid bolaamphiphiles either alone or as a constituent of a multicomponent lipid membrane may prove to be facile building blocks for generating robust bioactive membrane-mimetic assemblies. We have previously reported the synthesis of asymmetric dialkyl phospholipid bolaamphiphiles that contain ester linked phosphatidylcholine and amine functionalities at opposite chain ends. In this report, we describe the synthesis of phospholipid bolaamphiphiles that are conjugated to biotin via the terminal amine with or without a poly(ethylene oxide) spacer arm of varying chain length. The behavior of biotinylated bolaamphiphiles as a self-assembled monolayer at an air-water interface was characterized by epi-fluorescence microscopy and revealed that domain structure and pi-A isotherms were substantially influenced by linker type and size. Substrate bound assemblies were produced by Langmuir-Blodgett deposition onto planar substrates coated with an avidin derivatized polyelectrolyte multilayer. Significantly, external reflectance infrared spectroscopy confirmed the fabrication of bolaamphiphile thin films that display extended stability in vitro.

  19. Membrane lipid unsaturation as physiological adaptation to animal longevity

    PubMed Central

    Naudí, Alba; Jové, Mariona; Ayala, Victòria; Portero-Otín, Manuel; Barja, Gustavo; Pamplona, Reinald

    2013-01-01

    The appearance of oxygen in the terrestrial atmosphere represented an important selective pressure for ancestral living organisms and contributed toward setting up the pace of evolutionary changes in structural and functional systems. The evolution of using oxygen for efficient energy production served as a driving force for the evolution of complex organisms. The redox reactions associated with its use were, however, responsible for the production of reactive species (derived from oxygen and lipids) with damaging effects due to oxidative chemical modifications of essential cellular components. Consequently, aerobic life required the emergence and selection of antioxidant defense systems. As a result, a high diversity in molecular and structural antioxidant defenses evolved. In the following paragraphs, we analyze the adaptation of biological membranes as a dynamic structural defense against reactive species evolved by animals. In particular, our goal is to describe the physiological mechanisms underlying the structural adaptation of cellular membranes to oxidative stress and to explain the meaning of this adaptive mechanism, and to review the state of the art about the link between membrane composition and longevity of animal species. PMID:24381560

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

    SciTech Connect

    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 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. Lastly, the results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes.

  1. 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

  2. Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by 2H solid-state NMR spectroscopy.

    PubMed

    Vogel, Alexander; Schröder, Thomas; Lange, Christian; Huster, Daniel

    2007-12-01

    Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca2+ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca2+-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and 2H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the 2H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of -0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.

  3. Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

    PubMed Central

    Tunuguntla, Ramya; Bangar, Mangesh; Kim, Kyunghoon; Stroeve, Pieter; Ajo-Franklin, Caroline M.; Noy, Aleksandr

    2013-01-01

    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 biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we 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 the 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. PMID:24047990

  4. Lipid composition affects the rate of photosensitized dissipation of cross-membrane diffusion potential on liposomes

    PubMed Central

    Ytzhak, Shany; Wuskell, Joseph P.; Loew, Leslie M.; Ehrenberg, Benjamin

    2010-01-01

    Hydrophobic or amphiphilic tetrapyrrole sensitizers are taken up by cells and are usually located in cellular lipid membranes. Singlet oxygen is photogenerated by the sensitizer and it diffuses in the membrane and causes oxidative damage to membrane components. This damage can occur to membrane lipids and to membrane-localized proteins. Depolarization of the Nernst electric potential on cells’ membranes has been observed in cellular photosensitization, but it was not established whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells’ membranes and to their death. In this work we studied the effect of liposomes’ lipid composition on the kinetics of hematoporphyrin-photosensitized dissipation of K+-diffusion electric potential that was generated across the membranes. We employed an electrochromic voltage-sensitive spectroscopic probe that possesses a high fluorescence signal response to the potential. We found a correlation between the structure and unsaturation of lipids and the leakage of the membrane, following photosensitization. As the extent of non-conjugated unsaturation of the lipids is increased from 1 to 6 double bonds, the kinetics of depolarization become faster. We also found that the kinetics of depolarization is affected by the percentage of the unsaturated lipids in the liposome: as the fraction of the unsaturated lipids increases the leakage trough the membrane is enhanced. When liposomes are composed of a lipid mixture similar to that of natural membranes and photosensitization is being carried out under usual photodynamic therapy (PDT) conditions, photodamage to the lipids is not likely to cause enhanced permeability of ions through the membrane, which would have been a mechanism that leads to cell death. PMID:20536150

  5. Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

    SciTech Connect

    Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani; Fox, Daniel A.; Sim, Adelene Y.L.; Doniach, Sebastian; Lesley, Scott A.

    2009-10-21

    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

  6. Anomalous and normal diffusion of proteins and lipids in crowded lipid membranes.

    PubMed

    Javanainen, Matti; Hammaren, Henrik; Monticelli, Luca; Jeon, Jae-Hyung; Miettinen, Markus S; Martinez-Seara, Hector; Metzler, Ralf; Vattulainen, Ilpo

    2013-01-01

    Lateral diffusion plays a crucial role in numerous processes that take place in cell membranes, yet it is quite poorly understood in native membranes characterized by, e.g., domain formation and large concentration of proteins. In this article, we use atomistic and coarse-grained simulations to consider how packing of membranes and crowding with proteins affect the lateral dynamics of lipids and membrane proteins. We find that both packing and protein crowding have a profound effect on lateral diffusion, slowing it down. Anomalous diffusion is observed to be an inherent property in both protein-free and protein-rich membranes, and the time scales of anomalous diffusion and the exponent associated with anomalous diffusion are found to strongly depend on packing and crowding. Crowding with proteins also has a striking effect on the decay rate of dynamical correlations associated with lateral single-particle motion, as the transition from anomalous to normal diffusion is found to take place at macroscopic time scales: while in protein-poor conditions normal diffusion is typically observed in hundreds of nanoseconds, in protein-rich conditions the onset of normal diffusion is tens of microseconds, and in the most crowded systems as large as milliseconds. The computational challenge which results from these time scales is not easy to deal with, not even in coarse-grained simulations. We also briefly discuss the physical limits of protein motion. Our results suggest that protein concentration is anything but constant in the plane of cell membranes. Instead, it is strongly dependent on proteins' preference for aggregation.

  7. Lipid raft detecting in membranes of live erythrocytes.

    PubMed

    Mikhalyov, Ilya; Samsonov, Andrey

    2011-07-01

    The fluorescent probe N-(BODIPY(®)-FL-propionyl)-neuraminosyl-GM(1) (BODIPY-GM(1)) was used to detect lipid rafts in living red blood cells (RBCs) membranes. The probe was detected with fluorescence video microscopy and was found to be uniformly distributed along plasma membrane at room temperature (23°C). At 4°C some probe clearly phase-separated to yield detectable bright spots that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some probe had clustered. Red-shifted fluorescence was the greatest at 4°C, intermediate at 23°C, and the smallest at 37°C. Treating the RBCs with methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates that the presence of cholesterol was essential for phase separation of the probe. Fluorometry experiments indicate that rafts exist at 23°C and at 37°C, even though the membrane appears to be uniform at the resolution of microscope. The distinct GM(1) patches distributed over entire membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and fluorescence microscopy, some rafts clearly exist at 37°C.

  8. More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes.

    PubMed

    Ros, Uris; García-Sáez, Ana J

    2015-06-01

    Pore-forming proteins (PFPs) punch holes in their target cell membrane to alter their permeability. Permeabilization of lipid membranes by PFPs has received special attention to study the basic molecular mechanisms of protein insertion into membranes and the development of biotechnological tools. PFPs act through a general multi-step mechanism that involves (i) membrane partitioning, (ii) insertion into the hydrophobic core of the bilayer, (iii) oligomerization, and (iv) pore formation. Interestingly, PFPs and membranes show a dynamic interplay. As PFPs are usually produced as soluble proteins, they require a large conformational change for membrane insertion. Moreover, membrane structure is modified upon PFPs insertion. In this context, the toroidal pore model has been proposed to describe a pore architecture in which not only protein molecules but also lipids are directly involved in the structure. Here, we discuss how PFPs and lipids cooperate and remodel each other to achieve pore formation, and explore new evidences of protein-lipid pore structures.

  9. Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

    SciTech Connect

    Schewkunow, Vitali; Sharma, Karan P.; Diez, Gerold; Klemm, Anna H.; Sharma, Pal C.; Goldmann, Wolfgang H.

    2008-02-08

    A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 deg. C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.

  10. Atomic-level description of protein-lipid interactions using an accelerated membrane model.

    PubMed

    Baylon, Javier L; Vermaas, Josh V; Muller, Melanie P; Arcario, Mark J; Pogorelov, Taras V; Tajkhorshid, Emad

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov.

  11. Engineering lipid structure for recognition of the liquid ordered membrane phase

    DOE PAGES

    Bordovsky, Stefan S.; Wong, Christopher S.; Bachand, George D.; ...

    2016-08-26

    The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Furthermore, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (Lo) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, we foundmore » that although the lipid tails can direct selective partitioning to the Lo phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (Ld). The PEG spacer can serve as a buffer to mute headgroup–membrane interactions and thus improve Lo phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the Lo phase.« less

  12. Engineering lipid structure for recognition of the liquid ordered membrane phase

    SciTech Connect

    Bordovsky, Stefan S.; Wong, Christopher S.; Bachand, George D.; Stachowiak, Jeanne C.; Sasaki, Darryl Y.

    2016-08-26

    The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Furthermore, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (Lo) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, we found that although the lipid tails can direct selective partitioning to the Lo phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (Ld). The PEG spacer can serve as a buffer to mute headgroup–membrane interactions and thus improve Lo phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the Lo phase.

  13. The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes.

    PubMed

    Mojumdar, E H; Kariman, Z; van Kerckhove, L; Gooris, G S; Bouwstra, J A

    2014-10-01

    The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.

  14. Activation of integrin α5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells.

    PubMed

    Sun, Xiaoli; Fu, Yi; Gu, Mingxia; Zhang, Lu; Li, Dan; Li, Hongliang; Chien, Shu; Shyy, John Y-J; Zhu, Yi

    2016-01-19

    Local flow patterns determine the uneven distribution of atherosclerotic lesions. Membrane lipid rafts and integrins are crucial for shear stress-regulated endothelial function. In this study, we investigate the role of lipid rafts and integrin α5 in regulating the inflammatory response in endothelial cells (ECs) under atheroprone versus atheroprotective flow. Lipid raft proteins were isolated from ECs exposed to oscillatory shear stress (OS) or pulsatile shear stress, and then analyzed by quantitative proteomics. Among 396 proteins redistributed in lipid rafts, integrin α5 was the most significantly elevated in lipid rafts under OS. In addition, OS increased the level of activated integrin α5 in lipid rafts through the regulation of membrane cholesterol and fluidity. Disruption of F-actin-based cytoskeleton and knockdown of caveolin-1 prevented the OS-induced integrin α5 translocation and activation. In vivo, integrin α5 activation and EC dysfunction were observed in the atheroprone areas of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, and knockdown of integrin α5 markedly attenuated EC dysfunction in partially ligated carotid arteries. Consistent with these findings, mice with haploinsufficency of integrin α5 exhibited a reduction of atherosclerotic lesions in the regions under atheroprone flow. The present study has revealed an integrin- and membrane lipid raft-dependent mechanotransduction mechanism by which atheroprone flow causes endothelial dysfunction.

  15. Ionic channels and nerve membrane lipids. Cholesterol-tetrodotoxin interaction.

    PubMed

    Villegas, R; Barnola, F V; Camejo, G

    1970-04-01

    Experiments were carried out to investigate possible interactions of tetrodotoxin (TTX) with lipid molecules isolated from nerve fiber plasma membranes of the squid Dosidicus gigas. TTX has a highly selective ability to block the channel normally used by Na(+) to cross the axolemma during nervous impulse conduction. In order to investigate the interaction each lipid sample was spread on 5 x 10(-7)M TTX and TTX-free 0.15 M NaCl solutions adjusted to pH 7.4 with 7 x 10(-3)M phosphate buffer. The surface pressure-area diagrams of the lipid monolayers revealed that TTX interacts only with cholesterol. The expansion of the cholesterol monolayers at 5 x 10(-7)M TTX was 2 A(2)/molecule at zero pressure for the experiments at 20 degrees C and 2.5 A(2)/molecule for those at 25 degrees C. Similar results were obtained in KCl subphases. The apparent dissociation constant of the cholesterol-TTX complex calculated from dose-response experiments is 2.6 x 10(-7)M. Experiments at pH 10.1 revealed that the zwitter ionic form of TTX is less active. Experiments with cholesterol derivatives (cholesteryl acetate, cholesterol methyl ether, cholestanol, and cholestanyl acetate) indicate that for the interaction with TTX a partial negatively charged group at C-3 and a double bond between C-5 and C-6 on the steroid nucleus are required. Tetrodonic acid, a biologically inactive derivative of TTX, does not interact with cholesterol. The results lead us to propose that cholesterol is part of the Na(+) channel.

  16. Exploiting Lipid Permutation Symmetry to Compute Membrane Remodeling Free Energies

    NASA Astrophysics Data System (ADS)

    Bubnis, Greg; Risselada, Herre Jelger; Grubmüller, Helmut

    2016-10-01

    A complete physical description of membrane remodeling processes, such as fusion or fission, requires knowledge of the underlying free energy landscapes, particularly in barrier regions involving collective shape changes, topological transitions, and high curvature, where Canham-Helfrich (CH) continuum descriptions may fail. To calculate these free energies using atomistic simulations, one must address not only the sampling problem due to high free energy barriers, but also an orthogonal sampling problem of combinatorial complexity stemming from the permutation symmetry of identical lipids. Here, we solve the combinatorial problem with a permutation reduction scheme to map a structural ensemble into a compact, nondegenerate subregion of configuration space, thereby permitting straightforward free energy calculations via umbrella sampling. We applied this approach, using a coarse-grained lipid model, to test the CH description of bending and found sharp increases in the bending modulus for curvature radii below 10 nm. These deviations suggest that an anharmonic bending term may be required for CH models to give quantitative energetics of highly curved states.

  17. Control of plasma membrane lipid homeostasis by the extended synaptotagmins

    PubMed Central

    Saheki, Yasunori; Bian, Xin; Schauder, Curtis M.; Sawaki, Yujin; Surma, Michal A.; Klose, Christian; Pincet, Frederic; Reinisch, Karin M.; De Camilli, Pietro

    2016-01-01

    Acute metabolic changes of plasma membrane (PM) lipids, such as those mediating signaling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the Extended-Synaptotagmins (E-Syts), ER proteins which function as PI(4,5)P2 and Ca2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro and such transfer requires Ca2+ and their SMP domain, a lipid-harboring module. Genome edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but display enhanced and sustained accumulation of PM diacylglycerol (DAG) upon PI(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syts-dependent ER-PM tethers in response to stimuli that cleave PI(4,5)P2 and elevate Ca2+ may help reverse accumulation of DAG in the PM by transferring it to the ER for metabolic recycling. PMID:27065097

  18. Forty five years with membrane phospholipids, phospholipases and lipid mediators: A historical perspective.

    PubMed

    Chap, Hugues

    2016-06-01

    Phospholipases play a key role in the metabolism of phospholipids and in cell signaling. They are also a very useful tool to explore phospholipid structure and metabolism as well as membrane organization. They are at the center of this review, covering a period starting in 1971 and focused on a number of subjects in which my colleagues and I have been involved. Those include determination of phospholipid asymmetry in the blood platelet membrane, biosynthesis of lysophosphatidic acid, biochemistry of platelet-activating factor, first attempts to define the role of phosphoinositides in cell signaling, and identification of novel digestive (phospho)lipases such as pancreatic lipase-related protein 2 (PLRP2) or phospholipase B. Besides recalling some of our contributions to those various fields, this review makes an appraisal of the impressive and often unexpected evolution of those various aspects of membrane phospholipids and lipid mediators. It is also the occasion to propose some new working hypotheses.

  19. On ripples and rafts: Curvature induced nanoscale structures in lipid membranes

    NASA Astrophysics Data System (ADS)

    Schmid, Friederike; Dolezel, Stefan; Lenz, Olaf; Meinhardt, Sebastian

    2014-03-01

    We develop an elastic theory that predicts the spontaneous formation of nanoscale structures in lipid bilayers which locally phase separate between two phases with different spontaneous monolayer curvature. The theory rationalizes in a unified manner the observation of a variety of nanoscale structures in lipid membranes: Rippled states in one-component membranes, lipid rafts in multicomponent membranes. Furthermore, we report on recent observations of rippled states and rafts in simulations of a simple coarse-grained model for lipid bilayers, which are compatible with experimental observations and with our elastic model.

  20. On Physical Properties of Tetraether Lipid Membranes: Effects of Cyclopentane Rings

    PubMed Central

    Chong, Parkson Lee-Gau; Ayesa, Umme; Prakash Daswani, Varsha; Hur, Ellah Chay

    2012-01-01

    This paper reviews the recent findings related to the physical properties of tetraether lipid membranes, with special attention to the effects of the number, position, and configuration of cyclopentane rings on membrane properties. We discuss the findings obtained from liposomes and monolayers, composed of naturally occurring archaeal tetraether lipids and synthetic tetraethers as well as the results from computer simulations. It appears that the number, position, and stereochemistry of cyclopentane rings in the dibiphytanyl chains of tetraether lipids have significant influence on packing tightness, lipid conformation, membrane thickness and organization, and headgroup hydration/orientation. PMID:23028246

  1. A Study of Lipid Bilayer Membrane Stability Using Precise Measurements of Specific Capacitance

    PubMed Central

    White, Stephen H.

    1970-01-01

    A method is described for measuring the specific capacitance (Cm) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10-2 M KCl show that Cm depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), Cm increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm2 with a time constant of ∼15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. Cm of final-state membranes depends upon applied voltage (Va) and obeys the equation Cm = C0 + βVa2 where Va ≃ VDC + VrmsAC. C0 and β depend upon temperature; C0 decreases linearly with temperature while β increases linearly. At 20°C, C0 = 0.559 ±0.01 (SD) μF/cm2 and β = 0.0123 ±0.0036 (SD) (μF/cm2)/(mv2) and at 34°C, C0 = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in Cm are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves. ImagesFigure 2 PMID:5489777

  2. Voltage- and Tension-Dependent Lipid Mobility in the Outer Hair Cell Plasma Membrane

    NASA Astrophysics Data System (ADS)

    Oghalai, John S.; Zhao, Hong-Bo; Kutz, J. Walter; Brownell, William E.

    2000-01-01

    The mechanism responsible for electromotility of outer hair cells in the ear is unknown but is thought to reside within the plasma membrane. Lipid lateral diffusion in the outer hair cell plasma membrane is a sigmoidal function of transmembrane potential and bathing media osmolality. Cell depolarization or hyposmotic challenge shorten the cell and reduce membrane fluidity by half. Changing the membrane tension with amphipathic drugs results in similar reductions. These dynamic changes in membrane fluidity represent the modulation of membrane tension by lipid-protein interactions. The voltage dependence may be associated with the force-generating motors that contribute to the exquisite sensitivity of mammalian hearing.

  3. A novel squarylium dye for monitoring oxidative processes in lipid membranes.

    PubMed

    Trusova, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

    2009-11-01

    A novel squaraine probe SQ-1 has been found to be appropriate for monitoring the peroxidation processes in membrane systems. Formation of free radicals was triggered by methemoglobin (metHb) or cytochrome c (cyt c) binding to the model lipid membranes composed of zwitterionic lipid phosphatidylcholine (PC) and anionic lipid cardiolipin (CL). Protein association with the lipid vesicles was followed by drastic quenching of SQ-1 fluorescence. The observed spectral changes were suppressed in the presence of free radical scavengers, butylated hydroxytoluene (BHT) and thiourea (TM) suggesting that SQ-1 decolorization can be attributed to its reactions with lipid radicals.

  4. Alterations in Lipid Levels of Mitochondrial Membranes Induced by Amyloid-β: A Protective Role of Melatonin

    PubMed Central

    Rosales-Corral, Sergio A.; Lopez-Armas, Gabriela; Cruz-Ramos, Jose; Melnikov, Valery G.; Tan, Dun-Xian; Manchester, Lucien C.; Munoz, Ruben; Reiter, Russel J.

    2012-01-01

    Alzheimer pathogenesis involves mitochondrial dysfunction, which is closely related to amyloid-β (Aβ) generation, abnormal tau phosphorylation, oxidative stress, and apoptosis. Alterations in membranal components, including cholesterol and fatty acids, their characteristics, disposition, and distribution along the membranes, have been studied as evidence of cell membrane alterations in AD brain. The majority of these studies have been focused on the cytoplasmic membrane; meanwhile the mitochondrial membranes have been less explored. In this work, we studied lipids and mitochondrial membranes in vivo, following intracerebral injection of fibrillar amyloid-β (Aβ). The purpose was to determine how Aβ may be responsible for beginning of a vicious cycle where oxidative stress and alterations in cholesterol, lipids and fatty acids, feed back on each other to cause mitochondrial dysfunction. We observed changes in mitochondrial membrane lipids, and fatty acids, following intracerebral injection of fibrillar Aβ in aged Wistar rats. Melatonin, a well-known antioxidant and neuroimmunomodulator indoleamine, reversed some of these alterations and protected mitochondrial membranes from obvious damage. Additionally, melatonin increased the levels of linolenic and n-3 eicosapentaenoic acid, in the same site where amyloid β was injected, favoring an endogenous anti-inflammatory pathway. PMID:22666620

  5. The effect of charged lipids on bacteriorhodopsin membrane reconstitution and its photochemical activities

    SciTech Connect

    Wang Zhen; Bai Jing; Xu Yuhong

    2008-07-11

    Bacteriorhodopsin (BR) was reconstituted into artificial lipid membrane containing various charged lipid compositions. The proton pumping activity of BR under flash and continuous illumination, proton permeability across membrane, as well as the decay kinetics of the photocycle intermediate M{sub 412} were studied. The results showed that lipid charges would significantly affect the orientation of BR inserted into lipid membranes. In liposomes containing anionic lipids, BRs were more likely to take natural orientation as in living cells. In neutral or positively charged liposomes, most BRs were reversely assembled, assuming an inside out orientation. Moreover, the lipids charges also affect BR's M intermediate kinetics, especially the slow component in M intermediate decay. The half-life M{sub 412s} increased significantly in BRs in liposomes containing cationic lipids, while decreased in those in anionic liposomes.

  6. Lipid membrane structure and dynamics in the presence of tamoxifen and antimicrobial peptides

    NASA Astrophysics Data System (ADS)

    Hebenstreit, Samuel; Khadka, Nawal; Pan, Jianjun

    2015-03-01

    Lipids are organic molecules composed of hydrophobic fatty acid tails and hydrophilic head groups that can form a multitude of structures, including lipid vesicles which provides an excellent model representing cell membranes. In this study, we examine the effects of antimicrobial peptides and drugs on lipid vesicles. Fourier transform infrared spectroscopy measurements are performed with and without the antimicrobial peptide. A change in absorbance corresponding to the wavenumber regimes associated with the stretching of the carbonyl and phosphate groups is observed. Also, a dye leakage assay is performed with vesicles composed of neutral and charged lipids. Calcein dye is enclosed within these vesicles in solution. Different concentrations of the active and inactive antimicrobial peptides, and tamoxifen are incubated with the vesicles. Concentration dependent dye leakage is determined by measuring fluorescence intensity before and after the addition of the peptides and tamoxifen. Different leakage behavior is observed for the active and inactive peptides, and the lipid composition of the vesicle is found to have a large effect. Supported by an NSF grant.

  7. Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress.

    PubMed

    Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca

    2007-01-01

    Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.

  8. Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress▿

    PubMed Central

    Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca

    2007-01-01

    Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ9 position. We expressed two sunflower (Helianthus annuus) oleate Δ12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp+ or Trp− strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains. PMID:17071783

  9. Method of fabricating lipid bilayer membranes on solid supports

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

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

  10. Mechanics of water pore formation in lipid membrane under electric field

    NASA Astrophysics Data System (ADS)

    Bu, Bing; Li, Dechang; Diao, Jiajie; Ji, Baohua

    2017-02-01

    Transmembrane water pores are crucial for substance transport through cell membranes via membrane fusion, such as in neural communication. However, the molecular mechanism of water pore formation is not clear. In this study, we apply all-atom molecular dynamics and bias-exchange metadynamics simulations to study the process of water pore formation under an electric field. We show that water molecules can enter a membrane under an electric field and form a water pore of a few nanometers in diameter. These water molecules disturb the interactions between lipid head groups and the ordered arrangement of lipids. Following the movement of water molecules, the lipid head groups are rotated and driven into the hydrophobic region of the membrane. The reorientated lipid head groups inside the membrane form a hydrophilic surface of the water pore. This study reveals the atomic details of how an electric field influences the movement of water molecules and lipid head groups, resulting in water pore formation.

  11. Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes*

    PubMed Central

    Yagi, Hiromasa; Conroy, Paul J.; Leung, Eleanor W. W.; Law, Ruby H. P.; Trapani, Joseph A.; Voskoboinik, Ilia; Whisstock, James C.; Norton, Raymond S.

    2015-01-01

    Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes. PMID:26306037

  12. In vivo cluster formation of nisin and lipid II is correlated with membrane depolarization.

    PubMed

    Tol, Menno B; Morales Angeles, Danae; Scheffers, Dirk-Jan

    2015-01-01

    Nisin and related lantibiotics kill bacteria by pore formation or by sequestering lipid II. Some lantibiotics sequester lipid II into clusters, which were suggested to kill cells through delocalized peptidoglycan synthesis. Here, we show that cluster formation is always concomitant with (i) membrane pore formation and (ii) membrane depolarization. Nisin variants that cluster lipid II kill L-form bacteria with similar efficiency, suggesting that delocalization of peptidoglycan synthesis is not the primary killing mechanism of these lantibiotics.

  13. Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

    SciTech Connect

    Wallace, E.; Dranow, D.; Laible, P. D.; Christensen, J.; Nollert, P.

    2011-01-01

    The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization

  14. 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.

  15. Amounts of phospholipids and cholesterol in lipid domains formed in intact lens membranes: methodology development and its application to studies of porcine lens membranes

    PubMed Central

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

    2015-01-01

    An electron paramagnetic resonance spin-labeling method has been developed that allows quantitative evaluation of the amounts of phospholipids and cholesterol in lipid domains of intact fiber-cell plasma membranes isolated from cortical and nuclear regions of eye lenses. The long term goal of this research is the assessment of organizational changes in human lens fiber cell membranes that occur with age and during cataract development. The measurements needed to be performed on lens membranes prepared from eyes of single donors and from single eyes. For these types of studies it is necessary to separate the age/cataract related changes from preparation/technique related changes. Human lenses differ not only because of age, but also because of the varying health histories of the donors. To solve these problems the sample-to-sample preparation/technique related changes were evaluated for cortical and nuclear lens membranes prepared from single porcine eyes. It was assumed that the differences due to the age (animals were two year old) and environmental conditions for raising these animals were minimal. Mean values and standard deviations from preparation/technique changes for measured amounts of lipids in membrane domains were calculated. Statistical analysis (Student's t test) of the data also allowed determining the differences of mean values which were statistically significant with P ≤ 0.05. These differences defined for porcine lenses will be used for comparison of amounts of lipids in domains in human lens membranes prepared from eyes of single donors and from single eyes. Greater separations will indicate that differences were statistically significant with (P ≤ 0.05) and that they came from different than preparation/technique sources. Results confirmed that in nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the

  16. Amounts of phospholipids and cholesterol in lipid domains formed in intact lens membranes: Methodology development and its application to studies of porcine lens membranes.

    PubMed

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

    2015-11-01

    An electron paramagnetic resonance spin-labeling method has been developed that allows quantitative evaluation of the amounts of phospholipids and cholesterol in lipid domains of intact fiber-cell plasma membranes isolated from cortical and nuclear regions of eye lenses. The long term goal of this research is the assessment of organizational changes in human lens fiber cell membranes that occur with age and during cataract development. The measurements needed to be performed on lens membranes prepared from eyes of single donors and from single eyes. For these types of studies it is necessary to separate the age/cataract related changes from preparation/technique related changes. Human lenses differ not only because of age, but also because of the varying health histories of the donors. To solve these problems the sample-to-sample preparation/technique related changes were evaluated for cortical and nuclear lens membranes prepared from single porcine eyes. It was assumed that the differences due to the age (animals were two year old) and environmental conditions for raising these animals were minimal. Mean values and standard deviations from preparation/technique changes for measured amounts of lipids in membrane domains were calculated. Statistical analysis (Student's t-test) of the data also allowed determining the differences of mean values which were statistically significant with P ≤ 0.05. These differences defined for porcine lenses will be used for comparison of amounts of lipids in domains in human lens membranes prepared from eyes of single donors and from single eyes. Greater separations will indicate that differences were statistically significant with (P ≤ 0.05) and that they came from different than preparation/technique sources. Results confirmed that in nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than

  17. Conversion of membrane lipid acyl groups to triacylglycerol and formation of lipid bodies upon nitrogen starvation in biofuel green algae Chlorella UTEX29.

    PubMed

    Goncalves, Elton C; Johnson, Jodie V; Rathinasabapathi, Bala

    2013-11-01

    Algal lipids are ideal biofuel sources. Our objective was to determine the contributors to triacylglycerol (TAG) accumulation and lipid body formation in Chlorella UTEX29 under nitrogen (N) deprivation. A fivefold increase in intracellular lipids following N starvation for 24 h confirmed the oleaginous characteristics of UTEX29. Ultrastructural studies revealed increased number of lipid bodies and decreased starch granules in N-starved cells compared to N-replete cells. Lipid bodies were observed as early as 3 h after N removal and plastids collapsed after 48 h of stress. Moreover, the identification of intracellular pyrenoids and differences in the expected nutritional requirements for Chlorella protothecoides (as UTEX29 is currently classified) led us to conduct a phylogenetic study using 18S and actin cDNA sequences. This indicated UTEX29 to be more phylogenetically related to Chlorella vulgaris. To investigate the fate of different lipids after N starvation, radiolabeling using ¹⁴C-acetate was used. A significant decrease in ¹⁴C-galactolipids and phospholipids matched the increase in ¹⁴C-TAG starting at 3 h of N starvation, consistent with acyl groups from structural lipids as sources for TAG under N starvation. These results have important implications for the identification of key steps controlling oil accumulation in N-starved biofuel algae and demonstrate membrane recycling during lipid body formation.

  18. Biophysical interactions with model lipid membranes: applications in drug discovery and drug delivery

    PubMed Central

    Peetla, Chiranjeevi; Stine, Andrew; Labhasetwar, Vinod

    2009-01-01

    The transport of drugs or drug delivery systems across the cell membrane is a complex biological process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug molecules. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid composition of cells and tissue in certain disease conditions may alter biophysical interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophysical interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery. PMID:19432455

  19. Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion.

    PubMed

    Schultz, Zachary D; Pazos, Ileana M; McNeil-Watson, Fraser K; Lewis, E Neil; Levin, Ira W

    2009-07-23

    Interactions between dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS), combined both as binary lipid bilayer assemblies and separately, under the influence of divalent Mg2+, a membrane bilayer fusogenic agent, are reported. Infrared vibrational spectroscopic analyses of the lipid acyl chain methylene symmetric stretching modes indicate that aggregates of the two phospholipid components exist as domains heterogeneously distributed throughout the binary bilayer system. In the presence of Mg2+, DPPS maintains an ordered orthorhombic subcell gel phase structure through the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The addition of Mg2+ induces a reorganization of the lipid domains in which the gel phase acyl chain planes rearrange from a hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC-d62 in the absence of Mg2+. On adding Mg2+, a uniform DPPS domain size is observed in the binary mixtures. In either the presence or absence of Mg2+, DPPC-d62 aggregates remain in a configuration for which microdomain sizes are not spectroscopically measurable. Analysis of the acyl chain methylene deformation modes for DPPC-d62 in the binary system suggests that clusters of the deuterated lipids are distributed throughout the DPPS matrix. Light scattering and fluorescence measurements indicate that Mg2+ induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. The structural reorganizations of the lipid microdomains within the DPPS-DPPC bilayer are interpreted in the context of current concepts regarding lipid bilayer fusion.

  20. Sodium pump molecular activity and membrane lipid composition in two disparate ectotherms, and comparison with endotherms.

    PubMed

    Turner, Nigel; Hulbert, A J; Else, Paul L

    2005-02-01

    Previous research has shown that the lower sodium pump molecular activity observed in tissues of ectotherms compared to endotherms, is largely related to the lower levels of polyunsaturates and higher levels of monounsaturates found in the cell membranes of ectotherms. Marine-based ectotherms, however, have very polyunsaturated membranes, and in the current study, we measured molecular activity and membrane lipid composition in tissues of two disparate ectothermic species, the octopus (Octopus vulgaris) and the bearded dragon lizard (Pogona vitticeps), to determine whether the high level of membrane polyunsaturation generally observed in marine-based ectotherms is associated with an increased sodium pump molecular activity relative to other ectotherms. Phospholipids from all tissues of the octopus were highly polyunsaturated and contained high concentrations of the omega-3 polyunsaturate, docosahexaenoic acid (22:6 (n-3)). In contrast, phospholipids from bearded dragon tissues contained higher proportions of monounsaturates and lower proportions of polyunsaturates. Sodium pump molecular activity was only moderately elevated in tissues of the octopus compared to the bearded dragon, despite the much greater level of polyunsaturation in octopus membranes. When the current data were combined with data for the ectothermic cane toad, a significant (P = 0.003) correlation was observed between sodium pump molecular activity and the content of 22:6 (n-3) in the surrounding membrane. These results are discussed in relation to recent work which shows a similar relationship in endotherms.

  1. Thermal stability of ladderane lipids as determined by hydrous pyrolysis

    USGS Publications Warehouse

    Jaeschke, A.; Lewan, M.D.; Hopmans, E.C.; Schouten, S.; Sinninghe, Damste J.S.

    2008-01-01

    Anaerobic ammonium oxidation (anammox) has been recognized as a major process resulting in loss of fixed inorganic nitrogen in the marine environment. Ladderane lipids, membrane lipids unique to anammox bacteria, have been used as markers for the detection of anammox in marine settings. However, the fate of ladderane lipids after sediment burial and maturation is unknown. In this study, anammox bacterial cell material was artificially matured by hydrous pyrolysis at constant temperatures ranging from 120 to 365 ??C for 72 h to study the stability of ladderane lipids during progressive dia- and catagenesis. HPLC-MS/MS analysis revealed that structural alterations of ladderane lipids already occurred at 120 ??C. At temperatures >140 ??C, ladderane lipids were absent and only more thermally stable products could be detected, i.e., ladderane derivatives in which some of the cyclobutane rings were opened. These diagenetic products of ladderane lipids were still detectable up to temperatures of 260 ??C using GC-MS. Thus, ladderane lipids are unlikely to occur in ancient sediments and sedimentary rocks, but specific diagenetic products of ladderane lipids will likely be present in sediments and sedimentary rocks of relatively low maturity (i.e., C31 hopane 22S/(22S + 22R) ratio 0.5). ?? 2008 Elsevier Ltd.

  2. The role played by lipids unsaturation upon the membrane interaction of the Helicobacter pylori HP(2-20) antimicrobial peptide analogue HPA3.

    PubMed

    Mereuta, Loredana; Luchian, Tudor; Park, Yoonkynung; Hahm, Kyung-Soo

    2009-02-01

    The HPA3 peptide is an analogue of the linear antimicrobial peptide, HP(2-20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein, able to interact with zwitterionic lipid membranes and generate pores. Herein we focused on the importance of the degree of unsaturation of lipid acyl chains on HPA3 peptide-membrane interactions. Electrophysiology experiments carried out in reconstituted lipid membranes formed from phosphatidylcholines with one (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - POPC) and two monounsaturated acyl chains (1,2-dioleoyl-sn-glycero-3-phosphocholine - DOPC) demonstrate that the lesser degree of the packing density of membrane lipids encountered in DOPC-based planar membranes greatly enhances the electric activity of pores created by the HPA3 peptide. Data derived from fluorescence spectroscopy experiments demonstrate that upon interaction with the bilayer, the HPA3 peptide translocates to the trans-side of the membrane. From the same experiments, we demonstrate that in the case of DOPC-based planar membranes, the net amount of HPA3 peptide which passes across the membrane and re-dissolves in the trans solution is almost 22% greater than POPC-based membranes. Such data further emphasize the modulatory role played by lipid acyl chain in determining antimicrobial peptides-lipids interactions, and demonstrate that small differences in unsaturation degree can impose a sizeable influence on HPA3 peptide activity.

  3. Multivalent Rab interactions determine tether-mediated membrane fusion

    PubMed Central

    Lürick, Anna; Gao, Jieqiong; Kuhlee, Anne; Yavavli, Erdal; Langemeyer, Lars; Perz, Angela; Raunser, Stefan; Ungermann, Christian

    2017-01-01

    Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes. PMID:27852901

  4. Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

    NASA Astrophysics Data System (ADS)

    Zauscher, Stefan; Hardy, Gregory; Alam, Munir; Shapter, Joseph

    2012-02-01

    Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.

  5. Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

    PubMed

    Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan

    2015-04-01

    Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62-sn-glycero-3-phosphoglycerol (dDPPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/Escherichia coli (E. coli) polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide-bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide-bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide-cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties.

  6. Interactions of amiodarone with model membranes and amiodarone-photoinduced peroxidation of lipids.

    PubMed

    Sautereau, A M; Tournaire, C; Suares, M; Tocanne, J F; Paillous, N

    1992-06-23

    The potent antiarrhythmic drug, amiodarone (AMIO) exhibits phototoxicity, which is thought to be related to its interaction with biological membranes. We report here a spectroscopic study of the interactions of this drug with phosphatidylglycerol (PG) and phosphatidylcholine (PC) liposomes used as membrane model systems. A linear increase in absorbance at 300 nm was observed with increasing addition of AMIO to dimyristoyl-DL-PC (DMPC) liposomes over all the drugs-lipid molar ratio (Ri)s tested. In contrast, in the dimyristoyl-DL-PG (DMPG) liposomes, there was a dramatic increase in absorbance at values of Ri above unity. Light scattering by DMPG liposomes at 350 nm increased with increasing AMIO concentration up to a Ri = 1, and then decreased with increasing drug concentration. Such changes were not observed with the DMPC liposomes. Moreover, addition of AMIO changed the fluorescence polarization rate of 1,6-diphenyl 1,3,5-hexatriene embedded in these liposomes. It reduced the rate below the phase transition temperature (Tt) of the lipid, but increased it above this temperature. These effects on the lipidic phases observed at low Ri were more pronounced on the DMPG than on the DMPC liposomes. The strong interactions of AMIO with phospholipids, especially the acidic ones, were confirmed by liposome size determinations. All these data strongly suggest that the drug was incorporated in the core of the lipid bilayers. Such a penetration would favor a drug-photoinduced peroxidation of lipids. Indeed, UV irradiation of AMIO-DOPG mixtures led to the disappearance of the unsaturated fatty acids of phospholipids, checked by gas chromatography measurements, which was correlated with the amount of oxygen consumed. This showed that AMIO did photosensitize phospholipid peroxidation.

  7. The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing

    PubMed Central

    Liu, Aizhuo; Sui, Dexin; Wu, Dianqing; Hu, Jian

    2016-01-01

    Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K’s activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies. PMID:28138522

  8. Theoretical analysis of hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

    PubMed Central

    Harroun, T A; Heller, W T; Weiss, T M; Yang, L; Huang, H W

    1999-01-01

    We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated protein-protein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 A without gramicidin to 32.7 A. In contrast, the same molar ratio of gramicidin in DLPC increases the PtP from 30.8 A to 32.1 A. Concurrently, x-ray in-plane scattering showed that the most probable nearest-neighbor separation between gramicidin channels was 26.8 A in DLPC, but reduced to 23.3 A in DMPC. In this paper we review the idea of hydrophobic matching in which the lipid bilayer deforms to match the hydrophobic surface of the embedded proteins. We use a simple elasticity theory, including thickness compression, tension, and splay terms to describe the membrane deformation. The energy of membrane deformation is compared with the energy cost of hydrophobic mismatch. We discuss the boundary conditions between a gramicidin channel and the lipid bilayer. We used a numerical method to solve the problem of membrane deformation profile in the presence of a high density of gramicidin channels and ran computer simulations of 81 gramicidin channels to find the equilibrium distributions of the channels in the plane of the bilayer. The simulations contain four parameters: bilayer thickness compressibility 1/B, bilayer bending rigidity Kc, the channel-bilayer mismatch Do, and the slope of the interface at the lipid-protein boundary s. B, Kc, and Do were experimentally measured; the only free parameter is s. The value of s is determined by the requirement that the theory produces the experimental values of bilayer thinning by gramicidin and the shift in the peak position of the in-plane scattering due to membrane-mediated channel

  9. Probing Peptide and Protein Insertion in a Biomimetic S-Layer Supported Lipid Membrane Platform

    PubMed Central

    Damiati, Samar; Schrems, Angelika; Sinner, Eva-Kathrin; Sleytr, Uwe B.; Schuster, Bernhard

    2015-01-01

    The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided. PMID:25633104

  10. A model for surface diffusion of trans-membrane proteins on lipid bilayers

    NASA Astrophysics Data System (ADS)

    Agrawal, Ashutosh; Steigmann, David J.

    2011-06-01

    The equilibrium theory of lipid membranes is modified to include the effects of a continuous distribution of trans-membrane proteins. These influence membrane shape and evolve in accordance with a diffusive balance law. The model is purely mechanical in the absence of the proteins. Conditions ensuring energy dissipation in the presence of diffusion are given and an example constitutive function is used to simulate the coupled inertia-less interplay between membrane shape and protein distribution. The work extends an earlier continuum theory of equilibrium configurations of composite lipid-protein membranes to accommodate surface diffusion.

  11. 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.

  12. Monte Carlo Simulation of Protein-Induced Lipid Demixing in a Membrane with Interactions Derived from Experiment

    PubMed Central

    Almeida, Paulo F.; Best, Alexis; Hinderliter, Anne

    2011-01-01

    Lipid domain formation induced by annexin was investigated in mixtures of phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (Chol), which were selected to mimic the inner leaflet of a eukaryotic plasma membrane. Annexins are ubiquitous and abundant cytoplasmic, peripheral proteins, which bind to membranes containing PS in the presence of calcium ions (Ca2+), but whose function is unknown. Prompted by indications of interplay between the presence of cholesterol in PS/PC mixtures and the binding of annexins, we used Monte Carlo simulations to investigate protein and lipid domain formation in these mixtures. The set of interaction parameters between lipids and proteins was assigned by matching experimental observables to corresponding variables in the calculations. In the case of monounsaturated phospholipids, the PS-PC and PC-Chol interactions are weakly repulsive. The interaction between protein and PS was determined based on experiments of annexin binding to PC/PS mixtures in the presence of Ca2+. Based on the proposal that PS and cholesterol form a complex in model membranes, a favorable PS-Chol interaction was postulated. Finally, protein-protein favorable interactions were also included, which are consistent with observations of large, two-dimensional, regular arrays of annexins on membranes. Those net interactions between pairs of lipids, proteins and lipids, and between proteins are all small, of the order of the average kinetic energy. We found that annexin a5 can induce formation of large PS domains, coincident with protein domains, but only if cholesterol is present. PMID:22004747

  13. Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers.

    PubMed

    Paxman, Jason; Hunt, Brady; Hallan, David; Zarbock, Samuel R; Woodbury, Dixon J

    2017-01-10

    Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol's effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC50 = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.

  14. Lipid membrane-assisted condensation and assembly of amphiphilic Janus particles

    SciTech Connect

    Chambers, Mariah; Mallory, Stewart Anthony; Malone, Heather; Gao, Yuan; Anthony, Stephen M.; Yi, Yi; Cacciuto, Angelo; Yu, Yan

    2016-01-01

    Amphiphilic Janus particles self-assemble into complex metastructures, but little is known about how their assembly might be modified by weak interactions with a nearby biological membrane surface. Here, we report an integrated experimental and molecular dynamics simulation study to investigate the self-assembly of amphiphilic Janus particles on a lipid membrane. We created an experimental system in which Janus particles are allowed to self-assemble in the same medium where zwitterionic lipids form giant unilamellar vesicles (GUVs). Janus particles spontaneously concentrated on the inner leaflet of the GUVs. They exhibited biased orientation and heterogeneous rotational dynamics as revealed by single particle rotational tracking. The combined experimental and simulation results show that Janus particles concentrate on the lipid membranes due to weak particle–lipid attraction, whereas the biased orientation of particles is driven predominantly by inter-particle interactions. Furthermore, this study demonstrates the potential of using lipid membranes to influence the self-assembly of Janus particles.

  15. Analysis and quantification of plant membrane lipids by thin-layer chromatography and gas chromatography.

    PubMed

    Wewer, Vera; Dörmann, Peter; Hölzl, Georg

    2013-01-01

    Galactolipids represent the predominant membrane lipid class in plants. In general, galactolipids are restricted to plastids, but during phosphate deficiency, they also accumulate in extraplastidial membranes. Two groups of plants can be distinguished based on the presence of a specific fatty acid, hexadecatrienoic acid (16:3), in chloroplast lipids. Plants that contain galactolipids with 16:3 acids are designated "16:3-plants"; the other group of plants which lack 16:3 contain mostly 18:3 in their galactolipids ("18:3-plants"). The methods in this chapter describe the extraction of membrane lipids from whole leaves, or from subcellular fractions, and their analysis via thin-layer chromatography (TLC) with different staining methods. Furthermore, a protocol for membrane lipid quantification is presented starting with the separation via TLC, transmethylation of the isolated lipids to fatty acid methyl esters, and their quantitative analysis via gas chromatography (GC).

  16. Communication: Activation energy of tension-induced pore formation in lipid membranes.

    PubMed

    Karal, Mohammad Abu Sayem; Yamazaki, Masahito

    2015-08-28

    Tension plays a vital role in pore formation in biomembranes, but the mechanism of pore formation remains unclear. We investigated the temperature dependence of the rate constant of constant tension (σ)-induced pore formation in giant unilamellar vesicles of lipid membranes using an experimental method we developed. By analyzing this result, we determined the activation energy (Ua) of tension-induced pore formation as a function of tension. A constant (U0) that does not depend on tension was found to contribute significantly to Ua. Analysis of the activation energy clearly indicated that the dependence of Ua on σ in the classical theory is correct, but that the classical theory of pore formation is not entirely correct due to the presence of U0. We can reasonably consider that U0 is a nucleation free energy to form a hydrophilic pre-pore from a hydrophobic pre-pore or a region with lower lateral lipid density. After obtaining U0, the evolution of a pre-pore follows a classical theory. Our data provide valuable information that help explain the mechanism of tension-induced pore formation in biomembranes and lipid membranes.

  17. Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary

    PubMed Central

    Heinrich, Michael; Tian, Aiwei; Esposito, Cinzia; Baumgart, Tobias

    2010-01-01

    Cellular organelle membranes maintain their integrity, global shape, and composition despite vigorous exchange among compartments of lipids and proteins during trafficking and signaling. Organelle homeostasis involves dynamic molecular sorting mechanisms that are far from being understood. In contrast, equilibrium thermodynamics of membrane mixing and sorting, particularly the phase behavior of binary and ternary model membrane mixtures and its coupling to membrane mechanics, is relatively well characterized. Elucidating the continuous turnover of live cell membranes, however, calls for experimental and theoretical membrane models enabling manipulation and investigation of directional mass transport. Here we introduce the phenomenon of curvature-induced domain nucleation and growth in membrane mixtures with fluid phase coexistence. Membrane domains were consistently observed to nucleate precisely at the junction between a strongly curved cylindrical (tube) membrane and a pipette-aspirated giant unilamellar vesicle. This experimental geometry mimics intracellular sorting compartments, because they often show tubular-vesicular membrane regions. Nucleated domains at tube necks were observed to present diffusion barriers to the transport of lipids and proteins. We find that curvature-nucleated domains grow with characteristic parabolic time dependence that is strongly curvature-dependent. We derive an analytical model that reflects the observed growth dynamics. Numerically calculated membrane shapes furthermore allow us to elucidate mechanical details underlying curvature-dependent directed lipid transport. Our observations suggest a novel dynamic membrane sorting principle that may contribute to intracellular protein and lipid sorting and trafficking. PMID:20368457

  18. Interaction of anionic phenylene ethynylene polymers with lipids: from membrane embedding to liposome fusion.

    PubMed

    Karam, Pierre; Hariri, Amani A; Calver, Christina F; Zhao, Xiaoyong; Schanze, Kirk S; Cosa, Gonzalo

    2014-09-09

    Here we report spectroscopic studies on the interaction of negatively charged, amphiphilic polyphenylene ethynylene (PPE) polymers with liposomes prepared either from negative, positive or zwitterionic lipids. Emission spectra of PPEs of 7 and 49 average repeat units bearing carboxylate terminated side chains showed that the polymer embeds within positively charged lipids where it exists as free chains. No interaction was observed between PPEs and negatively charged lipids. Here the polymer remained aggregated giving rise to broad emission spectra characteristic of the aggregate species. In zwitterionic lipids, we observed that the majority of the polymer remained aggregated yet a small fraction readily embedded within the membrane. Titration experiments revealed that saturation of zwitterionic lipids with polymer typically occurred at a polymer repeat unit to lipid mole ratio close to 0.05. No further membrane embedding was observed above that point. For liposomes prepared from positively charged lipids, saturation was observed at a PPE repeat unit to lipid mole ratio of ∼0.1 and liposome precipitation was observed above this point. FRET studies showed that precipitation was preceded by lipid mixing and liposome fusion induced by the PPEs. This behavior was prominent for the longer polymer and negligible for the shorter polymer at a repeat unit to lipid mole ratio of 0.05. We postulate that fusion is the consequence of membrane destabilization whereby the longer polymer gives rise to more extensive membrane deformation than the shorter polymer.

  19. The Effect of Lidocaine · HCl on the Fluidity of Native and Model Membrane Lipid Bilayers

    PubMed Central

    Park, Jun-Seop; Jung, Tae-Sang; Noh, Yang-Ho; Kim, Woo-Sung; Park, Won-Ick; Kim, Young-Soo; Chung, In-Kyo; Sohn, Uy Dong; Bae, Soo-Kyung

    2012-01-01

    The purpose of this study is to investigated the mechanism of pharmacological action of local anesthetic and provide the basic information about the development of new effective local anesthetics. Fluorescent probe techniques were used to evaluate the effect of lidocaine·HCl on the physical properties (transbilayer asymmetric lateral and rotational mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Lidocaine·HCl increased the bulk lateral and rotational mobility of neuronal and model membrane lipid bilayes, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. Lidocaine·HCl increased annular lipid fluidity in SPMV lipid bilayers. It also caused membrane proteins to cluster. The most important finding of this study is that there is far greater increase in annular lipid fluidity than that in lateral and rotational mobilities by lidocaine·HCl. Lidocaine·HCl alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that lidocaine, in addition to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membrane lipid. PMID:23269904

  20. Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of α-synuclein

    PubMed Central

    Brown, James W. P.; Ouberai, Myriam M.; Flagmeier, Patrick; Vendruscolo, Michele; Buell, Alexander K.; Sparr, Emma; Dobson, Christopher M.

    2016-01-01

    Intracellular α-synuclein deposits, known as Lewy bodies, have been linked to a range of neurodegenerative disorders, including Parkinson’s disease. α-Synuclein binds to synthetic and biological lipids, and this interaction has been shown to play a crucial role for both α-synuclein’s native function, including synaptic plasticity, and the initiation of its aggregation. Here, we describe the interplay between the lipid properties and the lipid binding and aggregation propensity of α-synuclein. In particular, we have observed that the binding of α-synuclein to model membranes is much stronger when the latter is in the fluid rather than the gel phase, and that this binding induces a segregation of the lipids into protein-poor and protein-rich populations. In addition, α-synuclein was found to aggregate at detectable rates only when interacting with membranes composed of the most soluble lipids investigated here. Overall, our results show that the chemical properties of lipids determine whether or not the lipids can trigger the aggregation of α-synuclein, thus affecting the balance between functional and aberrant behavior of the protein. PMID:27298346

  1. Membrane lipid profiles of coral responded to zinc oxide nanoparticle-induced perturbations on the cellular membrane.

    PubMed

    Tang, Chuan-Ho; Lin, Ching-Yu; Lee, Shu-Hui; Wang, Wei-Hsien

    2017-03-31

    Zinc oxide nanoparticles (nZnOs) released from popular sunscreens used during marine recreation apparently endanger corals; however, the known biological effects are very limited. Membrane lipids constitute the basic structural element to create cell a dynamic structure according to the circumstance. Nano-specific effects have been shown to mechanically perturb the physical state of the lipid membrane, and the cells accommodating the actions of nZnOs can be involved in the alteration of the membrane lipid composition. To gain insight into the effects of nanoparticles on coral, glycerophosphocholine (GPC) profiling of the coral Seriatopora caliendrum exposed to nZnOs was performed in this study. Increasing lyso-GPCs, docosapentaenoic acid-possessing GPCs and docosahexaenoic acid-possessing GPCs and decreasing arachidonic acid-possessing GPCs were the predominant changes responded to nZnO exposure in the coral. A backfilling of polyunsaturated plasmanylcholines was observed in the coral exposed to nZnO levels over a threshold. These changes can be logically interpreted as an accommodation to nZnOs-induced mechanical disturbances in the cellular membrane based on the biophysical properties of the lipids. Moreover, the coral demonstrated a difference in the changes in lipid profiles between intra-colonial functionally differentiated polyps, indicating an initial membrane composition-dependent response. Based on the physicochemical properties and physiological functions of these changed lipids, some chronic biological effects can be incubated once the coral receives long-term exposure to nZnOs.

  2. Biophysics of cell membrane lipids in cancer drug resistance: Implications for drug transport and drug delivery with nanoparticles.

    PubMed

    Peetla, Chiranjeevi; Vijayaraghavalu, Sivakumar; Labhasetwar, Vinod

    2013-11-01

    In this review, we focus on the biophysics of cell membrane lipids, particularly when cancers develop acquired drug resistance, and how biophysical changes in resistant cell membrane influence drug transport and nanoparticle-mediated drug delivery. Recent advances in membrane lipid research show the varied roles of lipids in regulating membrane P-glycoprotein function, membrane trafficking, apoptotic pathways, drug transport, and endocytic functions, particularly endocytosis, the primary mechanism of cellular uptake of nanoparticle-based drug delivery systems. Since acquired drug resistance alters lipid biosynthesis, understanding the role of lipids in cell membrane biophysics and its effect on drug transport is critical for developing effective therapeutic and drug delivery approaches to overcome drug resistance. Here we discuss novel strategies for (a) modulating the biophysical properties of membrane lipids of resistant cells to facilitate drug transport and regain endocytic function and (b) developing effective nanoparticles based on their biophysical interactions with membrane lipids to enhance drug delivery and overcome drug resistance.

  3. A new Monte Carlo method for investigating geometrical structures of lipid membranes with atomistic detail

    NASA Astrophysics Data System (ADS)

    Cheng, Sara; Qiu, Liming; Cheng, K.; Vaughn, Mark

    2011-10-01

    The distribution statistics of the surface area, volume and voids of lipid molecules are important parameters to characterize the structures of self-assembling lipid membranes. Traditional methods are mostly based on various assumptions of the thickness of the lipid membrane and the volumes of certain types of lipid molecules. However, those methods usually lead to an over- or underestimation of the average surface area of lipid molecules when compared to the experimental results of the pure lipid systems. We developed a new Monte Carlo method that is able to estimate the distributions and averages of surface area, volume and void space of the lipid molecules in the absence and presence of proteins of the MD simulation results of lipid membranes at the atomistic scale. We successfully validated our new method on an ordered hard-sphere system and on a phospholipid/cholesterol binary lipid system, all with known structural parameters. Using this new method, the structural perturbation of the conformal annular lipids in close proximity to the embedded protein in a lipid/protein system will also be presented.

  4. Lipid flip-flop in binary membranes composed of phosphatidylserine and phosphatidylcholine.

    PubMed

    Brown, Krystal L; Conboy, John C

    2013-12-05

    The kinetics and thermodynamics of lipid flip-flop in bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were studied using sum-frequency vibrational spectroscopy. The kinetics of DSPC and DPPS flip-flop were examined as a function of temperature and bilayer composition. The rate of DSPC flip-flop did not exhibit any significant dependence on bilayer composition while the rate of DPPS flip-flop was inversely dependent on the mole fraction of DPPS. The transition-state thermodynamics for DSPC and DPPS lipids in these mixed bilayers were determined in order to identify the energetic impact of the phosphatidylserine headgroup on lipid flip-flop. The thermodynamics for the DSPC component remained statistically identical to bilayers composed entirely of DSPC. The activation energy for the DPPS component showed a linear correlation with the mole fraction of DPPS for all bilayer compositions. The enthalpy and entropy for DPPS flip-flop did not increase linearly with the fraction of DPPS but did directly correlate with the molecular area. The DPPS component also exhibited enthalpy-entropy compensation which suggests that lipid hydration may play a significant role in membrane dynamics.

  5. Lipid Replacement Therapy: a natural medicine approach to replacing damaged lipids in cellular membranes and organelles and restoring function.

    PubMed

    Nicolson, Garth L; Ash, Michael E

    2014-06-01

    Lipid Replacement Therapy, the use of functional oral supplements containing cell membrane phospholipids and antioxidants, has been used to replace damaged, usually oxidized, membrane glycerophospholipids that accumulate during aging and in various clinical conditions in order to restore cellular function. This approach differs from other dietary and intravenous phospholipid interventions in the composition of phospholipids and their defense against oxidation during storage, ingestion, digestion and uptake as well as the use of protective molecules that noncovalently complex with phospholipid micelles and prevent their enzymatic and bile disruption. Once the phospholipids have been taken in by transport processes, they are protected by several natural mechanisms involving lipid receptors, transport and carrier molecules and circulating cells and lipoproteins until their delivery to tissues and cells where they can again be transferred to intracellular membranes by specific and nonspecific transport systems. Once delivered to membrane sites, they naturally replace and stimulate removal of damaged membrane lipids. Various chronic clinical conditions are characterized by membrane damage, mainly oxidative but also enzymatic, resulting in loss of cellular function. This is readily apparent in mitochondrial inner membranes where oxidative damage to phospholipids like cardiolipin and other molecules results in loss of trans-membrane potential, electron transport function and generation of high-energy molecules. Recent clinical trials have shown the benefits of Lipid Replacement Therapy in restoring mitochondrial function and reducing fatigue in aged subjects and patients with a variety of clinical diagnoses that are characterized by loss of mitochondrial function and include fatigue as a major symptom. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

  6. UV-Visible and Infrared Methods for Investigating Lipid-Rhodopsin Membrane Interactions

    PubMed Central

    Brown, Michael F.

    2017-01-01

    Summary Experimental UV-visible and Fourier transform infrared (FTIR) spectroscopic methods are described for characterizing lipid-protein interactions for the example of rhodopsin in a membrane bilayer environment. The combined use of FTIR and UV-visible difference spectroscopy monitors the structural and functional changes during rhodopsin activation. Such studies investigate how membrane lipids stabilize the various rhodopsin photoproducts, analogous to mutating the protein. Interpretation of the results entails a non-specific flexible surface model for explaining the role of membrane lipid-protein interactions in biological functions. PMID:22976026

  7. Membrane Permeabilization Induced by Sphingosine: Effect of Negatively Charged Lipids

    PubMed Central

    Jiménez-Rojo, Noemi; Sot, Jesús; Viguera, Ana R.; Collado, M. Isabel; Torrecillas, Alejandro; Gómez-Fernández, J.C.; Goñi, Félix M.; Alonso, Alicia

    2014-01-01

    Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease. PMID:24940775

  8. LM cell growth and membrane lipid adaptation to sterol structure.

    PubMed

    Rujanavech, C; Silbert, D F

    1986-06-05

    Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.

  9. Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes.

    PubMed Central

    May, S; Harries, D; Ben-Shaul, A

    2000-01-01

    The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic. PMID:11023883

  10. Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes

    PubMed Central

    Hajj, Bassam; De Reguardati, Sophie; Hugonin, Loïc; Le Pioufle, Bruno; Osaki, Toshihisa; Suzuki, Hiroaki; Takeuchi, Shoji; Mojzisova, Halina; Chauvat, Dominique; Zyss, Joseph

    2009-01-01

    Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed. PMID:19948120

  11. Measuring lipid packing of model and cellular membranes with environment sensitive probes.

    PubMed

    Sezgin, Erdinc; Sadowski, Tomasz; Simons, Kai

    2014-07-15

    The extent of lipid packing is one of the key physicochemical features of biological membranes and is involved in many membrane processes. Polarity sensitive fluorescent probes are commonly used tools to measure membrane lipid packing in both artificial and biological membranes. In this paper, we have systematically compared eight different probes to measure membrane lipid ordering. We investigated how these probes behave in small unilamellar liposomes, phase-separated giant unilamellar vesicles, cell-derived giant plasma membrane vesicles, and live cells. We have tested the order sensitivity of a variety of measurable parameters, including generalized polarization, peak shift, or intensity shift. We also investigated internalization and photostability of the probes to assess probe potential for time-lapse live cell imaging. These results provide a catalogue of properties to facilitate the choice of probe according to need.

  12. Chemotherapy drugs form ion pores in membranes due to physical interactions with lipids.

    PubMed

    Ashrafuzzaman, Mohammad; Tseng, Chih-Yuan; Duszyk, Marek; Tuszynski, Jack A

    2012-12-01

    We demonstrate the effects on membrane of the tubulin-binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug-lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion-flowing pores across membranes. Discrete pore current-time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other's proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide-induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug-lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases.

  13. Arabidopsis chloroplast lipid transport protein TGD2 disrupts membranes and is part of a large complex.

    PubMed

    Roston, Rebecca; Gao, Jinpeng; Xu, Changcheng; Benning, Christoph

    2011-06-01

    In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.

  14. Dynamical Clustering and a Mechanism for Raft-like Structures in a Model Lipid Membrane

    PubMed Central

    Starr, Francis W.; Hartmann, Benedikt; Douglas, Jack F.

    2014-01-01

    We use molecular dynamics simulations to examine the dynamical heterogeneity of a model single-component lipid membrane using a coarse-grained representation of lipid molecules. This model qualitatively reproduces the known phase transitions between disordered, ordered, and gel membrane phases, and the phase transitions are accompanied by significant changes in the nature of the lipid dynamics. In particular, lipid diffusion in the liquid-ordered phase is hindered by the transient trapping of molecules by their neighbors, similar to the dynamics of a liquid approaching its glass transition. This transient molecular caging gives rise to two distinct mobility groups within a single-component membrane: lipids that are transiently trapped, and lipids with displacements on the scale of the intermolecular spacing. Most significantly, lipids within these distinct mobility states spatially segregate, creating transient “islands” of enhanced mobility having a size and time scale compatible with lipid “rafts,” dynamical structures thought to be important for cell membrane function. Although the dynamic lipid clusters that we observe do not themselves correspond to rafts (which are more complex, multicomponent structures), we hypothesize that such rafts may develop from the same universal mechanism, explaining why raft-like regions should arise, regardless of lipid structural or compositional details. These clusters are strikingly similar to the dynamical clusters found in glass-forming fluids, and distinct from phase-separation clusters. Further examination shows that mobile lipid clusters can be dissected into smaller clusters of cooperatively rearranging molecules. The geometry of these clusters can be understood in the context of branched equilibrium polymers, related to the statistics percolation theory. We discuss how these dynamical structures relate to a range observations on the dynamics of lipid membranes. PMID:24695573

  15. Chemotherapy Drugs Thiocolchicoside and Taxol Permeabilize Lipid Bilayer Membranes by Forming Ion Pores

    NASA Astrophysics Data System (ADS)

    Ashrafuzzaman, Md; Duszyk, M.; Tuszynski, J. A.

    2011-12-01

    We report ion channel formation by chemotherapy drugs: thiocolchicoside (TCC) and taxol (TXL) which primarily target tubulin but not only. For example, TCC has been shown to interact with GABAA, nuclear envelope and strychnine-sensitive glycine receptors. TXL interferes with the normal breakdown of microtubules inducing mitotic block and apoptosis. It also interacts with mitochondria and found significant chemotherapeutic applications for breast, ovarian and lung cancer. In order to better understand the mechanisms of TCC and TXL actions, we examined their effects on phospholipid bilayer membranes. Our electrophysiological recordings across membranes constructed in NaCl aqueous phases consisting of TCC or TXL under the influence of an applied transmembrane potential (V) indicate that both molecules induce stable ion flowing pores/channels in membranes. Their discrete current versus time plots exhibit triangular shapes which is consistent with a spontaneous time-dependent change of the pore conductance in contrast to rectangular conductance events usually induced by ion channels. These events exhibit conductance (~0.01-0.1 pA/mV) and lifetimes (~5-30 ms) within the ranges observed in e.g., gramicidin A and alamethicin channels. The channel formation probability increases linearly with TCC/TXL concentration and V and is not affected by pH (5.7 - 8.4). A theoretical explanation on the causes of chemotherapy drug induced ion pore formation and the pore stability has also been found using our recently discovered binding energy between lipid bilayer and the bilayer embedded ion channels using gramicidin A channels as tools. This picture of energetics suggests that as the channel forming agents approach to the lipids on bilayer the localized charge properties in the constituents of both channel forming agents (e.g., chemotherapy drugs in this study) and the lipids determine the electrostatic drug-lipid coupling energy through screened Coulomb interactions between the drug

  16. Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

    PubMed

    Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

    2016-04-19

    Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

  17. Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity.

    PubMed

    López-Pérez, Luis; Martínez-Ballesta, María Del Carmen; Maurel, Christophe; Carvajal, Micaela

    2009-03-01

    Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H(+)-ATPase. However, there was no effect of NaCl on water permeability (P(f)) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H(+)-ATPase activity, thereby helping to reduce partially the Na(+) concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H(+)-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.

  18. Effect of Hypoxia on the Calcium and Magnesium Content, Lipid Peroxidation Level, and Ca2+-ATPase Activity of Syncytiotrophoblast Plasma Membranes from Placental Explants

    PubMed Central

    Chiarello, Delia I.; Benzo, Zully; Piñero, Sandy; Botana, Desirée; Abad, Cilia

    2014-01-01

    In the current study the possible relationship between the Ca2+/Mg2+ ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca2+-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca2+ content, diminished their Ca2+-ATPase activity, and kept their Mg2+ content unchanged. Membranes preincubated with different concentrations of Ca2+ increased their Ca2+ content without changes in their Mg2+ content. There is a direct relationship between Ca2+ content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca2+ content and Ca2+-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg2+ showed a higher Mg2+ content without changing their lipid peroxidation and Ca2+-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca2+-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca2+ content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg2+ might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals. PMID:25180187

  19. Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains.

    PubMed

    Carquin, Mélanie; D'Auria, Ludovic; Pollet, Hélène; Bongarzone, Ernesto R; Tyteca, Donatienne

    2016-04-01

    The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicolson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decades, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (>min vs s) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryot es to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.

  20. Recent progress on lipid lateral heterogeneity in plasma membranes: from rafts to submicrometric domains

    PubMed Central

    Carquin, Mélanie; D'Auria, Ludovic; Pollet, Hélène; Bongarzone, Ernesto R.; Tyteca, Donatienne

    2016-01-01

    The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution. PMID:26738447

  1. The modified fluorescence based vesicle fluctuation spectroscopy technique for determination of lipid bilayer bending properties.

    PubMed

    Drabik, Dominik; Przybyło, Magda; Chodaczek, Grzegorz; Iglič, Aleš; Langner, Marek

    2016-02-01

    Lipid bilayer is the main constitutive element of biological membrane, which confines intracellular space. The mechanical properties of biological membranes may be characterized by various parameters including membrane stiffness or membrane bending rigidity, which can be measured using flicker noise spectroscopy. The flicker noise spectroscopy exploits the spontaneous thermal undulations of the membrane. The method is based on the quantitative analysis of a series of microscopic images captured during thermal membrane fluctuations. Thus, measured bending rigidity coefficient depends on the image quality as well as the selection of computational tools for image processing and mathematical model used. In this work scanning and spinning disc confocal microscopies were used to visualize fluctuating membranes of giant unilamellar vesicles. The bending rigidity coefficient was calculated for different acquisition modes, using different fluorescent probes and different image processing methods. It was shown that both imaging approaches gave similar bending coefficient values regardless of acquisition time. Using the developed methodology the effect of fluorescent probe type and aqueous phase composition on the value of the membrane bending rigidity coefficient was measured. Specifically it was found that the bending rigidity coefficient of DOPC bilayer in water is smaller than that determined for POPC membrane. It has been found that the POPC and DOPC bending rigidities coefficient in sucrose solution was lower than that in water. Fluorescence imaging makes possible the quantitative analysis of membrane mechanical properties of inhomogeneous membrane.

  2. Spherical Nanoparticle Supported Lipid Bilayers for the Structural Study of Membrane Geometry-Sensitive Molecules

    PubMed Central

    Kim, Edward Y.; Briley, Nicole E.; Tyndall, Erin R.; Xu, Jie; Li, Conggang; Ramamurthi, Kumaran S.; Flanagan, John M.; Tian, Fang

    2015-01-01

    Many essential cellular processes including endocytosis and vesicle trafficking require alteration of membrane geometry. These changes are usually mediated by proteins that can sense and/or induce membrane curvature. Using spherical nanoparticle supported lipid bilayers (SSLBs), we characterize how SpoVM, a bacterial development factor, interacts with differently curved membranes by magic angle spinning solid-state NMR. Our results demonstrate that SSLBs are an effective system for structural and topological studies of membrane geometry-sensitive molecules. PMID:26488086

  3. Mechano-capacitive properties of polarized membranes and the application to conductance measurements of lipid membrane patches

    NASA Astrophysics Data System (ADS)

    Zecchi, Karis A.; Mosgaard, Lars D.; Heimburg, Thomas

    2017-01-01

    Biological membranes are capacitors that can be charged by applying an electric field across the membrane. The charges on the capacitor exert a force on the membrane that leads to electrostriction, i.e., a thinning of the membrane. This effect is especially strong close to chain melting transitions. A consequence is voltage induced pore formation in the lipid membrane. Since the force is quadratic in voltage, negative and positive voltages have an identical influence on the physics of symmetric membranes. This is not the case for a membrane with an asymmetry leading to a permanent electric polarization. Positive and negative voltages of identical magnitude lead to different physical properties. Such an asymmetry can originate from a lipid composition that is different on the two monolayers of the membrane, or from membrane curvature. The latter effect is called flexoelectricity. It was investigated in detail by A.G. Petrov in the recent decades. As a consequence of permanent polarization, the membrane capacitor is discharged at a voltage different from zero. This leads to interesting electrical phenomena such as outward or inward rectification of membrane permeability. The changes in current-voltage relationships are consistent with the known magnitude of the flexoelectric effect.

  4. Lipid intermediates in membrane fusion: formation, structure, and decay of hemifusion diaphragm.

    PubMed Central

    Kozlovsky, Yonathan; Chernomordik, Leonid V; Kozlov, Michael M

    2002-01-01

    Lipid bilayer fusion is thought to involve formation of a local hemifusion connection, referred to as a fusion stalk. The subsequent fusion stages leading to the opening of a fusion pore remain unknown. The earliest fusion pore could represent a bilayer connection between the membranes and could be formed directly from the stalk. Alternatively, fusion pore can form in a single bilayer, referred to as hemifusion diaphragm (HD), generated by stalk expansion. To analyze the plausibility of stalk expansion, we studied the pathway of hemifusion theoretically, using a recently developed elastic model. We show that the stalk has a tendency to expand into an HD for lipids with sufficiently negative spontaneous splay, (~)J(s)< 0. For different experimentally relevant membrane configurations we find two characteristic values of the spontaneous splay. (~)J*(s) and (~)J**(s), determining HD dimension. The HD is predicted to have a finite equilibrium radius provided that the spontaneous splay is in the range (~)J**(s)< (~)J(s)<(~)J*(s), and to expand infinitely for (~)J(s)<(~)J**(s). In the case of common lipids, which do not fuse spontaneously, an HD forms only under action of an external force pulling the diaphragm rim apart. We calculate the dependence of the HD radius on this force. To address the mechanism of fusion pore formation, we analyze the distribution of the lateral tension emerging in the HD due to the establishment of lateral equilibrium between the deformed and relaxed portions of lipid monolayers. We show that this tension concentrates along the HD rim and reaches high values sufficient to rupture the bilayer and form the fusion pore. Our analysis supports the hypothesis that transition from a hemifusion to a fusion pore involves radial expansion of the stalk. PMID:12414697

  5. Responses of lipid membranes of taste sensor to astringent and pungent substances.

    PubMed

    Iiyama, S; Toko, K; Matsuno, T; Yamafuji, K

    1994-02-01

    Astringent substances and pungent substances were studied using a multichannel taste sensor with lipid membranes. The electric-potential pattern constructed of eight outputs from the membranes has information of taste quality and intensity. Pungent substances, such as capsaicin, piperine and allyl isothiocyanate, had no effect on the membrane potentials of the lipid membranes. On the other hand, astringent substances such as tannic acid, catechin, gallic acid and chlorogenic acid changed the potentials remarkably. A principal component analysis of the patterns in electric potential changes caused by the taste substances revealed the astringency is located between bitterness and sourness.

  6. Study of sweet taste evaluation using taste sensor with lipid/polymer membranes.

    PubMed

    Habara, Masaaki; Ikezaki, Hidekazu; Toko, Kiyoshi

    2004-07-15

    The higher sensitivity for sweeteners can be achieved by newly developed lipid/polymer membranes. The membrane is composed of lipids such as phosphoric acid di-n-hexadecyl ester and tetradodecylammoniumbromid, and a plasticizer, dioctyl phenylphosphonate. As a result of changing electric charge of the membrane surface, the newly developed membrane shows 5-10 times higher sensitivity for sucrose than the conventional ones. We also applied the sensor to other sugars such as sugar alcohol which is used as alternative sweetness or food additives. The experimental results of other sweeteners relatively correspond to human sensory evaluation, though the sensitivity for some sugars need to be improved.

  7. 3D-Membrane Stacks on Supported Membranes Composed of Diatom Lipids Induced by Long-Chain Polyamines.

    PubMed

    Gräb, Oliver; Abacilar, Maryna; Daus, Fabian; Geyer, Armin; Steinem, Claudia

    2016-10-04

    Long-chain polyamines (LCPAs) are intimately involved in the biomineralization process of diatoms taking place in silica deposition vesicles being acidic compartments surrounded by a lipid bilayer. Here, we addressed the question whether and how LCPAs interact with lipid membranes composed of glycerophospholipids and glyceroglycolipids mimicking the membranes of diatoms and higher plants. Solid supported lipid bilayers and monolayers containing the three major components that are unique in diatoms and higher plants, i.e., monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), were prepared by spreading small unilamellar vesicles. The integrity of the membranes was investigated by fluorescence microscopy and atomic force microscopy showing continuous flat bilayers and monolayers with small protrusions on top of the membrane. The addition of a synthetic polyamine composed of 13 amine groups separated by a propyl spacer (C3N13) results in flat but three-dimensional membrane stacks within minutes. The membrane stacks are connected with the underlying membrane as verified by fluorescence recovery after photobleaching experiments. Membrane stack formation was found to be independent of the lipid composition; i.e., neither glyceroglycolipids nor negatively charged lipids were required. However, the formation process was strongly dependent on the chain length of the polyamine. Whereas short polyamines such as the naturally occurring spermidine, spermine, and the synthetic polyamines C3N4 and C3N5 do not induce stack formation, those containing seven and more amine groups (C3N7, C3N13, and C3N18) do form membrane stacks. The observed stack formation might have implications for the stability and expansion of the silica deposition vesicle during valve and girdle band formation in diatoms.

  8. Interaction of alpha-latroinsectotoxin from Latrodectus mactans venom with bilayer lipid membranes.

    PubMed

    Shatursky OYa; Pashkov, V N; Bulgacov, O V; Grishin, E V

    1995-01-26

    alpha-Latroinsectotoxin (LIT) from Latrodectus mactans venom increased the conductance of bilayer lipid membranes (BLM) by inducing channel like activity. The channels formed had a maximal single channel conductance of 5 pS in 10 mM CaCl2 solution. This process occurred more rapidly in symmetrical 10 mM CaCl2 solution than in equimolar KCl or NaCl. The LIT induced conductance showed pronounced rectification, that was dependent upon the face of the BLM to which the LIT was applied. This suggests that the LIT molecules incorporate into the bilayer lipid membrane in an oriented manner. The ion channels formed in bilayer phospholipid membrane by LIT are cation selective. The permeability of divalent cations decreased in the order Ba2+ > Ca2+ > Mg2+ > Cd2+ > Zn2+ (Zn2+ and Cd2+ blocked effectively LIT channels with the ratio of Ca2+trans and Cd2+cis or Zn2+cis of 1:1). Selectivity of LIT to monovalent cations was not high and was Ca2+ sensitive. Our data suggest that LIT has at least two Ca(2+)-binding sites, a high affinity site and low one (pK of binding is 2.4). As a result, the binding kinetics of Ca2+ with the toxin shows a high positive cooperativity (Hill coefficient, (h) = 5.95) and that dimerization might be a prerequisite to channel formation. Temperature dependence of conductance of LIT treated lipid bilayers in 100 mM KCl and 10 mM CaCl2 solutions was also determined: 18.9 +/- 2.11 kJ/mol and 28.537 +/- 1.678 kJ/mol, respectively.

  9. Insulin Receptor Processing and Lipid Composition of Erythrocyte Membrane in Patients with Hyperlipidemia.

    PubMed

    Masella, R.; Cantafora, A.; Maffì, D.; Volpe, R.; Ginnetti, M.G.; Ricci, G.; Mace, N.L.; Buxton, G.M.; Peterson, S.W.

    1995-08-01

    The aim of this study was to determine whether the common forms of dyslipidemia could affect either the lipid composition or insulin receptor processing (down-regulation) of erythrocytes. The study included 22 patients with type IIa hypercholesterolemia, 15 patients with type IV hypertriglyceridemia and 12 patients with type IIb hyperlipidemia. Ten normolipidemic subjects were used as controls. Their erythrocyte membranes were analyzed for lipid composition and insulin receptor down-regulation. The results show that all the hyperlipidemias investigated were characterized by significant increases in the cholesterol to phospholipid molar ratio (0.56 +/- 0.08 in controls and 1.11 +/- 0.13, 1.09 +/- 0.14, 1.04 +/- 0.15, p < 0.001, in types IIa, IIb and IV, respectively). Surface insulin receptors of type IIa and IIb patients did not appear to down-regulate when compared to normal subjects, but rather up-regulated (+65.2% in controls, -1.0% and -8.7%, p < 0.001, in type IIa and IIb patients, respectively). Patients with type IV hypertriglyceridemia showed a residual capacity for insulin receptor internalization (10.7% down-regulation). Membranes of all the patients contained a higher proportion of phosphatidylethanolamine; the molar ratio of sphingomyelin to phosphatidylcholine was significantly higher in types IIb than in controls (1.22 +/- 0.11 and 1.12 +/- 0.10, p < 0.05, respectively); all the patients showed a lower content of polyunsaturated fatty acids in the major glycerophospholipid classes. However, type IV hypertriglyceridemics showed less variations, especially in the phosphatidylserine fraction. These results indicate that the alterations in lipoprotein pattern may affect both the lipid membrane equilibria and the processing ability of surface insulin receptors. Copyright 1995 S. Karger AG, Basel

  10. Gramicidin Alters the Lipid Compositions of Liquid-Ordered and Liquid-Disordered Membrane Domains

    NASA Astrophysics Data System (ADS)

    Hassan-Zadeh, Ebrahim; Huang, Juyang

    2012-10-01

    The effects of adding 1 mol % of gramicidin A to the well-known DOPC/DSPC/cholesterol lipid mixtures were investigated. 4-component giant unilamellar vesicles (GUV) were prepared using our recently developed Wet-Film method. The phase boundary of liquid-ordered and liquid-disordered (Lo-Ld) coexisting region was determined using video fluorescence microscopy. We found that if cares were not taken, light-induced domain artifacts could significantly distort the measured phase boundary. After testing several fluorescence dyes, we found that the emission spectrum of Nile Red is quite sensitive to membrane composition. By fitting the Nile Red emission spectra at the phase boundary to the spectra in the Lo-Ld coexisting region, the thermodynamic tie-lines were determined. As an active component of lipid membranes, gramicidin not only partitions favorably into the liquid-disordered (Ld) phase, it also alters the phase boundary and thermodynamic tie-lines. Even at as low as 1 mol %, gramicidin decreases the cholesterol mole fraction of Ld phase and increases the area of Lo phase.

  11. Imidazolium-Based Lipid Analogues and Their Interaction with Phosphatidylcholine Membranes.

    PubMed

    Wang, Da; de Jong, Djurre H; Rühling, Andreas; Lesch, Volker; Shimizu, Karina; Wulff, Stephanie; Heuer, Andreas; Glorius, Frank; Galla, Hans-Joachim

    2016-12-06

    4,5-Dialkylated imidazolium lipid salts are a new class of lipid analogues showing distinct biological activities. The potential effects of the imidazolium lipids on artificial lipid membranes and the corresponding membrane interactions was analyzed. Therefore, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was employed to create an established lipid monolayer model and a bilayer membrane. Mixed monolayers of DPPC and 4,5-dialkylimidazolium lipids differing by their alkyl chain length (C7, C11, and C15) were characterized by surface pressure-area (π-A) isotherms using a Wilhelmy film balance in combination with epifluorescence microscopy. Monolayer hysteresis for binary mixtures was examined by recording triplicate consecutive compression-expansion cycles. The lipid miscibility and membrane stability of DPPC/imidazolium lipids were subsequently evaluated by the excess mean molecular area (ΔA(ex)) and the excess Gibbs free energy (ΔG(ex)) of mixing. Furthermore, the thermotropic behavior of mixed liposomes of DPPC/imidazolium lipids was investigated by differential scanning calorimetry (DSC). The C15-imidazolium lipid (C15-IMe·HI) forms a thermodynamically favored and kinetically reversible Langmuir monolayer with DPPC and exhibits a rigidification effect on both DPPC monolayer and bilayer structures at low molar fractions (X ≤ 0.3). However, the incorporation of the C11-imidazolium lipid (C11-IMe·HI) causes the formation of an unstable and irreversible Langmuir-Gibbs monolayer with DPPC and disordered DPPC liposomes. The C7-imidazolium lipid (C7-IMe·HI) displays negligible membrane activity. To better understand these results on a molecular level, all-atom molecular dynamics (MD) simulations were performed. The simulations yield two opposing molecular mechanisms governing the different behavior of the three imidazolium lipids: a lateral ordering effect and a free volume/stretching effect. Overall, our study provides the first evidence that the membrane

  12. Differential effect of plant lipids on membrane organization: specificities of phytosphingolipids and phytosterols.

    PubMed

    Grosjean, Kevin; Mongrand, Sébastien; Beney, Laurent; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2015-02-27

    The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains.

  13. Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

    PubMed Central

    Grison, Magali S.; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M.

    2015-01-01

    Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. PMID:25818623

  14. Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

    PubMed

    Grison, Magali S; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M

    2015-04-01

    Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.

  15. Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH.

    PubMed

    Dainese, Enrico; De Fabritiis, Gianni; Sabatucci, Annalaura; Oddi, Sergio; Angelucci, Clotilde Beatrice; Di Pancrazio, Chiara; Giorgino, Toni; Stanley, Nathaniel; Del Carlo, Michele; Cravatt, Benjamin F; Maccarrone, Mauro

    2014-02-01

    Lipid composition is expected to play an important role in modulating membrane enzyme activity, in particular if the substrates are themselves lipid molecules. A paradigmatic case is FAAH (fatty acid amide hydrolase), an enzyme critical in terminating endocannabinoid signalling and an important therapeutic target. In the present study, using a combined experimental and computational approach, we show that membrane lipids modulate the structure, subcellular localization and activity of FAAH. We report that the FAAH dimer is stabilized by the lipid bilayer and shows a higher membrane-binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate AEA (anandamide). Additionally, co-localization of cholesterol, AEA and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH.

  16. Altered membrane lipid composition and functional parameters of circulating cells in cockles (Cerastoderma edule) affected by disseminated neoplasia.

    PubMed

    Le Grand, Fabienne; Soudant, Philippe; Marty, Yanic; Le Goïc, Nelly; Kraffe, Edouard

    2013-01-01

    Membrane lipid composition and morpho-functional parameters were investigated in circulating cells of the edible cockle (Cerastoderma edule) affected by disseminated neoplasia (neoplastic cells) and compared to those from healthy cockles (hemocytes). Membrane sterol levels, phospholipid (PL) class and subclass proportions and their respective fatty acid (FA) compositions were determined. Morpho-functional parameters were evaluated through total hemocyte count (THC), mortality rate, phagocytosis ability and reactive oxygen species (ROS) production. Both morpho-functional parameters and lipid composition were profoundly affected in neoplastic cells. These dedifferentiated cells displayed higher THC (5×), mortality rate (3×) and ROS production with addition of carbonyl cyanide m-chloro phenylhydrazone (1.7×) but lower phagocytosis ability (½×), than unaffected hemocytes. Total PL amounts were higher in neoplastic cells than in hemocytes (12.3 and 5.1 nmol×10(-6) cells, respectively). However, sterols and a particular subclass of PL (plasmalogens; 1-alkenyl-2-acyl PL) were present in similar amounts in both cell type membranes. This led to a two times lower proportion of these membrane lipid constituents in neoplastic cells when compared to hemocytes (20.5% vs. 42.1% of sterols in total membrane lipids and 21.7% vs. 44.2% of plasmalogens among total PL, respectively). Proportions of non-methylene interrupted FA- and 20:1n-11-plasmalogen molecular species were the most impacted in neoplastic cells when compared to hemocytes (⅓× and ¼×, respectively). These changes in response to this leukemia-like disease in bivalves highlight the specific imbalance of plasmalogens and sterols in neoplastic cells, in comparison to the greater stability of other membrane lipid components.

  17. Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions.

    PubMed

    Boggs, J M

    1986-01-01

    The large number of different membrane lipids with various structural modifications and properties and the characteristic lipid composition of different types of membranes suggest that different lipids have specific functions in the membrane. Many of the varying properties of lipids with different polar head groups and in different ionization states can be attributed to the presence of interactive or repulsive forces between the head groups in the bilayer. The interactive forces are hydrogen bonds between hydrogen bond donating groups such as --P--OH,--OH, and--NH3+ and hydrogen bond accepting groups such as --P--O- and --COO-. These interactions increase the lipid phase transition temperature and can account for the tendency of certain lipids to go into the hexagonal phase and the dependence of this tendency on the pH and ionization state of the lipid. The presence or absence of these interactions can also affect the penetration of hydrophobic substances into the bilayer, including hydrophobic residues of membrane proteins. Evidence for this suggestion has been gathered from studies of the myelin basic protein, a water-soluble protein with a number of hydrophobic residues. In this way the lipid composition can affect the conformation and activity of membrane proteins. Since hydrogen-bonding interactions depend on the ionization state of the lipid, they can be altered by changes in the environment which affect the pK of the ionizable groups. The formation of the hexagonal phase or inverted micelles, the conformation and activity of membrane proteins, and other functions mediated by lipids could thus be regulated in this way.

  18. Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.

    PubMed Central

    Mustardy, L; Los, D A; Gombos, Z; Murata, N

    1996-01-01

    There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes. Images Fig. 1 Fig. 2 Fig. 3 PMID:11607709

  19. 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.

  20. Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

    PubMed

    Verstraeten, Sandra V; Jaggers, Grayson K; Fraga, Cesar G; Oteiza, Patricia I

    2013-11-01

    Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.

  1. Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen.

    PubMed

    Alsop, Richard J; Armstrong, Clare L; Maqbool, Amna; Toppozini, Laura; Dies, Hannah; Rheinstädter, Maikel C

    2015-06-28

    There is increasing evidence that common drugs, such as aspirin and ibuprofen, interact with lipid membranes. Ibuprofen is one of the most common over the counter drugs in the world, and is used for relief of pain and fever. It interacts with the cyclooxygenase pathway leading to inhibition of prostaglandin synthesis. From X-ray diffraction of highly oriented model membranes containing between 0 and 20 mol% ibuprofen, 20 mol% cholesterol, and dimyristoylphosphatidylcholine (DMPC), we present evidence for a non-specific interaction between ibuprofen and cholesterol in lipid bilayers. At a low ibuprofen concentrations of 2 mol%, three different populations of ibuprofen molecules were found: two in the lipid head group region and one in the hydrophobic membrane core. At higher ibuprofen concentrations of 10 and 20 mol%, the lamellar bilayer structure is disrupted and a lamellar to cubic phase transition was observed. In the presence of 20 mol% cholesterol, ibuprofen (at 5 mol%) was found to be expelled from the membrane core and reside solely in the head group region of the bilayers. 20 mol% cholesterol was found to stabilize lamellar membrane structure and the formation of a cubic phase at 10 and 20 mol% ibuprofen was suppressed. The results demonstrate that ibuprofen interacts with lipid membranes and that the interaction is strongly dependent on the presence of cholesterol.

  2. Removal of lipopolysaccharide from protein solution using nanostructured porous supports bearing lipid membranes

    NASA Astrophysics Data System (ADS)

    Wakita, Masa-aki

    2013-11-01

    Polymeric lipid membranes of N-octadecylchitosan, which consists of 70 mol% of 2-(octadecylamino)-2-deoxy- d-glucopyranose, 17 mol% of 2-amino-2-deoxy- d-glucopyranose, and 13 mol% of 2-acetamido-2-deoxy- d-glucopyranose, were covalently immobilized to carboxylated porous supports composed of chitosan and used for the adsorption of pyrogenic lipopolysaccharide. When human serum albumin solution, including 5 mg mL-1 of albumin and 5.6 ng mL-1 of lipopolysaccharide, was passed through a column packed with the resulting porous supports bearing lipid membranes assembled in nanoscale, lipopolysaccharide was removed to as low as a detection limit of 0.020 ng mL-1 with a quantitative recovery of protein. On the other hand, in the case of directly N-octadecylated porous supports having cationic and hydrophobic ligands which are not assembled as lipid membranes, lipopolysaccharide could not be removed to the detection limit and protein recovery was lower than the porous supports bearing lipid membranes. The difference above as well as difference from conventional adsorbents suggested that the selectivity was attributable to an interaction between the cationic lipid membranes of N-octadecylchitosan and lipopolysaccharide as well as protein. The porous supports bearing lipid membranes were stable in 0.5 M NaOH and 0.1 M HCl at ambient temperature. Considering the confirmed excellent selectivity and chemical stability, their practical use as separation media in the pharmaceutical manufacturing can be expected.

  3. Lipid binding and membrane penetration of polymyxin B derivatives studied in a biomimetic vesicle system.

    PubMed Central

    Katz, Marina; Tsubery, Haim; Kolusheva, Sofiya; Shames, Alex; Fridkin, Mati; Jelinek, Raz

    2003-01-01

    Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing D-instead of L-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane. PMID:12848621

  4. Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution.

    PubMed

    Fujita, Akikazu; Cheng, Jinglei; Fujimoto, Toyoshi

    2010-04-01

    An important goal of membrane biology is to define the local heterogeneity of membrane lipid composition. Here we describe a quantitative electron microscopic method that enables the localization of specific membrane lipids at the nanometer scale. The method involves freezing cells rapidly to halt the molecular motion, physically stabilizing membrane molecules in the freeze-fracture replica by the deposition of evaporated platinum and carbon layers and labeling with specific probes for electron microscopic observation. Lipids in both the outer and inner membrane leaflets can thus be labeled, and their distributions can be analyzed quantitatively by statistical methods. A major advantage of this method is that it does not require the expression of artificial probes. Therefore, this method can be applied to any cell in vitro or in vivo, and the whole procedure can be completed in 1-2 d.

  5. Computer simulations of the diffusion of Na+ and Cl- ions across POPC lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Salih, Rangeen; Matthai, C. C.

    2017-03-01

    We have carried out molecular dynamics simulations using NAMD to study the diffusivity of Na and Cl ions across a POPC lipid bilayer membrane. We show that an imbalance of positively and negatively charged ions on either side of the membrane leads to the diffusion of ions and water molecules. We considered the cases of both weak and very strong charge imbalance across the membrane. The diffusion coefficients of the ions have been determined from the mean square displacements of the particles as a function of time. We find that for strong electrochemical gradients, both the Na and Cl ions diffuse rapidly through pores in the membrane with diffusion coefficients up to ten times larger than in water. Rather surprisingly, we found that although the Na ions are the first to begin the permeation process due to the lower potential barrier that they experience compared to the Cl ions, the latter complete the permeation across the barrier more quickly due to their faster diffusion rates.

  6. Properties of Membranes Derived from the Total Lipids Extracted from the Human Lens Cortex and Nucleus

    PubMed Central

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

    2013-01-01

    Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses. PMID:23438364

  7. Coupling between pore formation and phase separation in charged lipid membranes

    NASA Astrophysics Data System (ADS)

    Himeno, Hiroki; Ito, Hiroaki; Higuchi, Yuji; Hamada, Tsutomu; Shimokawa, Naofumi; Takagi, Masahiro

    2015-12-01

    We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids. We observed the membrane morphologies by fluorescent and confocal laser microscopy in lipid mixtures consisting of a neutral unsaturated lipid [dioleoylphosphatidylcholine (DOPC)], a neutral saturated lipid [dipalmitoylphosphatidylcholine (DPPC)], a charged unsaturated lipid [dioleoylphosphatidylglycerol (DOP G(-)) ], a charged saturated lipid [dipalmitoylphosphatidylglycerol (DPP G(-)) ], and cholesterol (Chol). In binary mixtures of neutral DOPC-DPPC and charged DOPC -DPP G(-) , spherical vesicles were formed. On the other hand, pore formation was often observed with GUVs consisting of DOP G(-) and DPPC. In a DPPC-DPPG(-) -Chol ternary mixture, pore-formed vesicles were also frequently observed. The percentage of pore-formed vesicles increased with the DPP G(-) concentration. Moreover, when the head group charges of charged lipids were screened by the addition of salt, pore-formed vesicles were suppressed in both the binary and ternary charged lipid mixtures. We discuss the mechanisms of pore formation in charged lipid mixtures and the relationship between phase separation and the membrane morphology. Finally, we reproduce the results seen in experimental systems by using coarse-grained molecular dynamics simulations.

  8. Mapping of Membrane Lipid Order in Root Apex Zones of Arabidopsis thaliana

    PubMed Central

    Zhao, Xiaoyu; Zhang, Xiran; Qu, Yanli; Li, Ruili; Baluška, František; Wan, Yinglang

    2015-01-01

    In this study, we used the fluorescence probe, Di-4-ANEPPDHQ, to map the distribution of membrane lipid order in the apical region of Arabidopsis roots. The generalized polarization (GP) value of Di-4-ANEPPDHQ-stained roots indicated the highest lipid order in the root transition zone (RTZ). The cortical cells have higher lipid order than the epidermal cells in same regions, while the developing root hairs show very prominent cell polarity with high lipid order in apical region. Moreover, the endosomes had lower lipid order than that of the plasma membrane (PM). Brefeldin A (BFA) treatment decreased the lipid order in both the plasma and endosomal membranes of epidermal cells in the RTZ. The lipid order of BFA-induced compartments became higher than that of the PM after BFA treatment in epidermal cells. Meanwhile, the polarly growing tips of root hairs did not show the same behavior. The lipid order of the PM remained unchanged, with higher values than that of the endosomes. This suggests that the lipid ordering in the PM was affected by recycling of endosomal vesicles in epidermal cells of the root apex transition zone but not in the root hairs of Arabidopsis. PMID:26734047

  9. [Effect of microwaves on bilayer lipid membranes: role of a membrane-forming hole in the Teflon film].

    PubMed

    Alekseev, S I; Ziskin, M S; Fesenko, E E

    2009-01-01

    The distributions of specific abcorption rate (SAR) and E-field in a membrane-forming hole of Teflon film and surrounding electrolyte were calculated for 0.9 GHz exposure. It was found that the specific absorption rate in the membrane-forming hole increased greatly with increasing thickness of the Teflon film, and electrolyte concentration and decreasing diameter of the hole. The previously demonstrated significant changes in the conductivity of modified bilayer lipid membranes induced by microwave exposure can be explained by a local increase in specific absorption rate and subsequent elevation of temperature in the membrane-forming hole of the Teflon film.

  10. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

    PubMed Central

    Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

    2012-01-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

  11. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains.

    PubMed

    Krager, Kimberly J; Sarkar, Mitul; Twait, Erik C; Lill, Nancy L; Koland, John G

    2012-10-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.

  12. Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis

    PubMed Central

    Aktas, Meriyem; Danne, Linna; Möller, Philip; Narberhaus, Franz

    2014-01-01

    Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930

  13. An ultrasensitive tool exploiting hydration dynamics to decipher weak lipid membrane-polymer interactions.

    PubMed

    Cheng, Chi-Yuan; Wang, Jia-Yu; Kausik, Ravinath; Lee, Ka Yee C; Han, Songi

    2012-02-01

    We introduce a newly developed tool, (1)H Overhauser Dynamic Nuclear Polarization (ODNP), to sensitively explore weak macromolecular interactions by site-specifically probing the modulation of the translational dynamics of hydration water at the interaction interface, in the full presence of bulk water. Here, ODNP is employed on an illustrative example of a membrane-active triblock copolymer, poloxamer 188 (P188), which is known to restore the integrity of structurally compromised cell membranes. We observe a distinct change in the translational dynamics of the hydration layer interacting with the lipid membrane surface and the bilayer-interior as P188 is added to a solution of lipid vesicles, but no measurable changes in the dynamics or structure of the lipid membranes. This study shows that hydration water is an integral constituent of a lipid membrane system, and demonstrates for the first time that the modulation of its translational diffusivity can sensitively report on weak polymer-membrane interactions, as well as mediate essential lipid membrane functions. ODNP holds much promise as a unique tool to unravel molecular interactions at interfaces even in the presence of bulk water under ambient conditions.

  14. Lipopolysaccharide-Induced Dynamic Lipid Membrane Reorganization: Tubules, Perforations, and Stacks

    PubMed Central

    Adams, Peter G.; Lamoureux, Loreen; Swingle, Kirstie L.; Mukundan, Harshini; Montaño, Gabriel A.

    2014-01-01

    Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na+ leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca2+ gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions. PMID:24896118

  15. Membrane topology of transmembrane proteins: determinants and experimental tools.

    PubMed

    Lee, Hunsang; Kim, Hyun

    2014-10-17

    Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.

  16. Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes

    NASA Astrophysics Data System (ADS)

    Fradin, Cécile; Satsoura, Dmitri; Andrews, David W.

    Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes — namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.

  17. A theoretical formalism for aggregation of peroxidized lipids and plasma membrane stability during photolysis.

    PubMed Central

    Busch, N A; Yarmush, M L; Toner, M

    1998-01-01

    The objective of this investigation was to examine, from a theoretical perspective, the mechanism underlying the lysis of plasma membranes by photoinduced, chemically mediated damage such as is found in photolysis. Toward this end, a model is presented which relates the membrane lifetime to the thermodynamic parameters of the membrane components based upon the kinetic theory of aggregate formation. The formalism includes a standard birth/death process for the formation of damaged membrane components (i.e., peroxidized lipids) as well as a terminating condensation process for the formation of aggregates of peroxidized plasma membrane lipids. Our theory predicts that 1) the membrane lifetime is inversely correlated with predicted rate of membrane damage; 2) an upper limit on the duration of membrane damage exists, above which the mean and variance of the membrane lifetime is independent of further membrane damage; and 3) both the mean and variance of the time of membrane lifetime distribution are correlated with the number of sites that may be damaged to form a single membrane defect. The model provides a framework to optimize the lysis of cell membranes by photodynamic therapy. PMID:9826616

  18. Randomization of membrane lipids in relation to transport system assembly in Escherichia coli.

    PubMed

    Thilo, L; Overath, P

    1976-01-27

    The distribution of newly synthesized lipid molecules in the pre-existing lipid phase of the membrane was studied in whole cells of the fatty acid requiring Escheria coli strain K1062. The fluorescence probe N-phenyl-1-naphthylamine revealed reversible lipid phase transitions in cells supplemented with cis-delta9-octadecenoate (transition temperature Tt = 14 degrees C; width of the transition deltaT = 13 degrees C) or trans-delta9-hexadecenoate (Tt = 27 degrees C; deltaT = 7 degrees C). Cells were first grown in the presence of cis-delta9-octadecenoate at 37 degrees C and subsequently for various periods in the presence of trans-delta9-hexadecenoate at 37 or 22 degrees C, i.e. above or below the transition of the newly formed lipids. Reproducible phase transitions with single, well-defined Tt values between 14 and 27 degrees C were observed under both conditions. Beta-Galactoside transport induced in a similar experiment before or during a change in the fatty acid composition showed a single change in activation energy at a temperature close to the lipid transition temperature, Tt. Starvation of cis-delta9-octadecenoate-supplemented cells for this fatty acid led to a gradual rise in the transition temperature, due to an increase in the percentage of saturated acyl chains in the membrane lipids. It is concluded that under all conditions investigated a mixed lipid phase composed of newly synthesized and pre-existing lipid molecules is formed in the membrane. Since conserved domains of newly synthesized lipids surrounding simultaneously formed transport proteins could not be demonstrated, the results do not support a membrane assembly mechanism proposed by N. Tsukagoshi and C. F. Fox [(1973), Biochemistry 12, 2822-2829]. It rather appears that newly formed lipid molecules are continuously released from their sites of synthesis into the lipid matrix by a rapid diffusion-controlled process.

  19. The Membrane and Lipids as Integral Participants in Signal Transduction: Lipid Signal Transduction for the Non-Lipid Biochemist

    ERIC Educational Resources Information Center

    Eyster, Kathleen M.

    2007-01-01

    Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction.…

  20. Lipid vesicular nanocarrier: Quick encapsulation efficiency determination and transcutaneous application.

    PubMed

    Zhang, Yibang; Ng, Weibeng; Feng, Xue; Cao, Fangying; Xu, Huaxi

    2017-01-10

    Nanoscale delivery systems have been widely investigated to overcome the penetration barrier of stratum corneum for effective transcutaneous application. The aim of this study is the development of effective vesicular formulations of ovalbumin and saponin which are able to promote penetration through the skin layers. Three kinds of vesicular formulations have been investigated as carriers, including liposomes, transfersomes and ethosomes, in which cholesterol and/or cationic lipid stearylamine are incorporated. The impact of membrane composition variations on the protein entrapment has been evaluated for each vesicle type. Formulations were characterized for particle size, polydispersity and encapsulation efficiency. The best formulations for each type of vesicle were subjected to in vivo transdermal immunization in mice. Among the three kinds of vesicular carrier, ethosomal nano carrier not only showed the best stability over a two months' storage, but also enabled the highest increase in the titer of serum antibody. In this regard, cationic nano-ethosomes can be considered as a promising vesicular carrier for transdermal vaccines. Meanwhile, we have developed a simple method to determine encapsulation efficiency of vesicular systems, which has potential application as a high throughput screening for vesicular formulations.

  1. (19)F NMR screening of unrelated antimicrobial peptides shows that membrane interactions are largely governed by lipids.

    PubMed

    Afonin, Sergii; Glaser, Ralf W; Sachse, Carsten; Salgado, Jesús; Wadhwani, Parvesh; Ulrich, Anne S

    2014-09-01

    Many amphiphilic antimicrobial peptides permeabilize bacterial membranes via successive steps of binding, re-alignment and/or oligomerization. Here, we have systematically compared the lipid interactions of two structurally unrelated peptides: the cyclic β-pleated gramicidin S (GS), and the α-helical PGLa. (19)F NMR was used to screen their molecular alignment in various model membranes over a wide range of temperatures. Both peptides were found to respond to the phase state and composition of these different samples in a similar way. In phosphatidylcholines, both peptides first bind to the bilayer surface. Above a certain threshold concentration they can re-align and immerse more deeply into the hydrophobic core, which presumably in