bilayer membrane fusion: Topics by WorldWideScience.org

Sample records for bilayer membrane fusion

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

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

2013-05-28

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

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Azusa Oshima

2017-09-01

Full Text Available The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs to control the fusion process. We combined large unilamellar vesicles (LUVs containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO2 substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.
The computational route from bilayer membranes to vesicle fusion

International Nuclear Information System (INIS)

Shillcock, Julian C; Lipowsky, Reinhard

2006-01-01

Biological membranes are examples of 'smart' materials whose properties and behaviour emerge from the propagation across many scales of the molecular characteristics of their constituents. Artificial smart materials, such as drug delivery vehicles and biosensors, often rely on modifying naturally occurring soft matter, such as polymers and lipid vesicles, so that they possess useful behaviour. However, the complexity of natural membranes, both in their static properties, exemplified in their phase behaviour, and in their dynamic properties, as in the kinetics of their formation and interactions, hinders their rational modification. Mesoscopic simulations, such as dissipative particle dynamics (DPD), allow in silico experiments to be easily and cheaply performed on complex, soft materials requiring as input only the molecular structure of the constituents at a coarse-grained level. They can therefore act as a guide to experimenters prior to performing costly assays. Additionally, mesoscopic simulations provide the only currently feasible window on the length- and timescales relevant to important biophysical processes such as vesicle fusion. We review here the development of computational models of bilayer membranes, and in particular the use of mesoscopic simulations to follow the molecular rearrangements that occur during membrane fusion
Viral membrane fusion

International Nuclear Information System (INIS)

Harrison, Stephen C.

2015-01-01

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

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

2015-05-15

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

Science.gov (United States)

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.
Effect of phospholipid metabolites on model membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Shragin, A.S.; Vasilenko, I.A.; Selishcheva, A.A.; Shvets, V.I.

1985-01-01

/sup 31/P-NMR spectroscopy and formation of fluorescent complexes between Tb/sup 3 +/ and dipicolinic acid were used to monitor liposome fusion and the effects of phospholipases C and D on the process. Phospholipase C was found highly efficient in initiating liposomal fusion, regardless of the phospholipid composition of the bilayer membranes. However, phospholipase D promoted liposomal fusion only in cases in which the membranes contained high concentrations of phospholipids incapable of forming bilayer membranes, such as phosphatidylethanolamine and cardiolipin. The mechanism of action of both enzymes in promoting liposomal fusion was ascribed to the generation of a metastable state in the membranes as a result of enzymatic formation of lipophilic metabolites 1,2-diacylglycerol and phosphatidic acid. The perturbation, or fluidity, of the liposomal membranes favored fusion on contact. 21 references, 4 figures.
Vesicle fusion observed by content transfer across a tethered lipid bilayer.

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Rawle, Robert J; van Lengerich, Bettina; Chung, Minsub; Bendix, Poul Martin; Boxer, Steven G

2011-10-19

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion*

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Lai, Alex L.; Tamm, Lukas K.

2010-01-01

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

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Zick, Michael; Stroupe, Christopher; Orr, Amy; Douville, Deborah; Wickner, William T

2014-01-01

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.
Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion.

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Ravoo, B J; Weringa, W D; Engberts, J B

2000-01-01

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se. Copyright 2000 Academic Press.
Biophysical characterization and membrane interaction of the two fusion loops of glycoprotein B from herpes simplex type I virus.

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Annarita Falanga

Full Text Available The molecular mechanism of entry of herpesviruses requires a multicomponent fusion system. Cell invasion by Herpes simplex virus (HSV requires four virally encoded glycoproteins: namely gD, gB and gH/gL. The role of gB has remained elusive until recently when the crystal structure of HSV-1 gB became available and the fusion potential of gB was clearly demonstrated. Although much information on gB structure/function relationship has been gathered in recent years, the elucidation of the nature of the fine interactions between gB fusion loops and the membrane bilayer may help to understand the precise molecular mechanism behind herpesvirus-host cell membrane fusion. Here, we report the first biophysical study on the two fusion peptides of gB, with a particular focus on the effects determined by both peptides on lipid bilayers of various compositions. The two fusion loops constitute a structural subdomain wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. When used together the two fusion loops have the ability to significantly destabilize the target membrane bilayer, notwithstanding their low bilayer penetration when used separately. These data support the model of gB fusion loops insertion into cholesterol enriched membranes.
Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

International Nuclear Information System (INIS)

Pszon-Bartosz, Kamila; Hansen, Jesper S.; Stibius, Karin B.; Groth, Jesper S.; Emneus, Jenny; Geschke, Oliver; Helix-Nielsen, Claus

2011-01-01

Research highlights: → We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). → Maximal fusion obtained was almost 150,000 porin insertions during 20 min. → Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10 5 FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm 2 within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.
Interaction of elaiophylin with model bilayer membrane

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Genova, J.; Dencheva-Zarkova, M.

2017-01-01

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

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Kozlovsky, Yonathan; Chernomordik, Leonid V; Kozlov, Michael M

2002-11-01

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)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.
Shear-Induced Membrane Fusion in Viscous Solutions

KAUST Repository

Kogan, Maxim

2014-05-06

Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We present evidence that shear forces in a viscous solution can induce lipid bilayer fusion. The fusion of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) liposomes is observed in Couette flow with shear rates above 3000 s-1 provided that the medium is viscous enough. Liposome samples, prepared at different viscosities using a 0-50 wt % range of sucrose concentration, were studied by dynamic light scattering, lipid fusion assays using Förster resonance energy transfer (FRET), and linear dichroism (LD) spectroscopy. Liposomes in solutions with 40 wt % (or more) sucrose showed lipid fusion under shear forces. These results support the hypothesis that under suitable conditions lipid membranes may fuse in response to mechanical-force- induced stress. © 2014 American Chemical Society.
Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

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Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.
Fusion of raft-like lipid bilayers operated by a membranotropic domain of the HSV-type I glycoprotein gH occurs through a cholesterol-dependent mechanism.

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Vitiello, Giuseppe; Falanga, Annarita; Petruk, Ariel Alcides; Merlino, Antonello; Fragneto, Giovanna; Paduano, Luigi; Galdiero, Stefania; D'Errico, Gerardino

2015-04-21

A wealth of evidence indicates that lipid rafts are involved in the fusion of the viral lipid envelope with the target cell membrane. However, the interplay between these sterol- and sphingolipid-enriched ordered domains and viral fusion glycoproteins has not yet been clarified. In this work we investigate the molecular mechanism by which a membranotropic fragment of the glycoprotein gH of the Herpes Simplex Virus (HSV) type I (gH625) drives fusion of lipid bilayers formed by palmitoyl oleoyl phosphatidylcholine (POPC)-sphingomyelin (SM)-cholesterol (CHOL) (1 : 1 : 1 wt/wt/wt), focusing on the role played by each component. The comparative analysis of the liposome fusion assays, Dynamic Light Scattering (DLS), spectrofluorimetry, Neutron Reflectivity (NR) and Electron Spin Resonance (ESR) experiments, and Molecular Dynamics (MD) simulations shows that CHOL is fundamental for liposome fusion to occur. In detail, CHOL stabilizes the gH625-bilayer association by specific interactions with the peptide Trp residue. The interaction with gH625 causes an increased order of the lipid acyl chains, whose local rotational motion is significantly hampered. SM plays only a minor role in the process, favoring the propagation of lipid perturbation to the bilayer inner core. The stiffening of the peptide-interacting bilayer leaflet results in an asymmetric perturbation of the membrane, which is locally destabilized thus favoring fusion events. Our results show that viral fusion glycoproteins are optimally suited to exert a high fusogenic activity on lipid rafts and support the relevance of cholesterol as a key player of membrane-related processes.
Engineering plant membranes using droplet interface bilayers.

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

2017-03-01

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

International Nuclear Information System (INIS)

Bokoch, Michael P.; Devadoss, Anando; Palencsar, Mariela S.; Burgess, James D.

2004-01-01

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented

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

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

2011-01-01

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

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

Science.gov (United States)

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. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.
Prevention of intra-abdominal adhesion by bi-layer electrospun membrane.

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

2013-06-04

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

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....
Computational Design of Multi-component Bio-Inspired Bilayer Membranes

Directory of Open Access Journals (Sweden)

Evan Koufos

2014-04-01

Full Text Available Our investigation is motivated by the need to design bilayer membranes with tunable interfacial and mechanical properties for use in a range of applications, such as targeted drug delivery, sensing and imaging. We draw inspiration from biological cell membranes and focus on their principal constituents. In this paper, we present our results on the role of molecular architecture on the interfacial, structural and dynamical properties of bio-inspired membranes. We focus on four lipid architectures with variations in the head group shape and the hydrocarbon tail length. Each lipid species is composed of a hydrophilic head group and two hydrophobic tails. In addition, we study a model of the Cholesterol molecule to understand the interfacial properties of a bilayer membrane composed of rigid, single-tail molecular species. We demonstrate the properties of the bilayer membranes to be determined by the molecular architecture and rigidity of the constituent species. Finally, we demonstrate the formation of a stable mixed bilayer membrane composed of Cholesterol and one of the phospholipid species. Our approach can be adopted to design multi-component bilayer membranes with tunable interfacial and mechanical properties. We use a Molecular Dynamics-based mesoscopic simulation technique called Dissipative Particle Dynamics that resolves the molecular details of the components through soft-sphere coarse-grained models and reproduces the hydrodynamic behavior of the system over extended time scales.
Formation of supported lipid bilayers of charged E. coli lipids on modified gold by vesicle fusion

Directory of Open Access Journals (Sweden)

Ileana F. Márquez

2017-01-01

Full Text Available We describe a simple way of fusing E. coli lipid vesicles onto a gold surface. Supported lipid bilayers on metal surfaces are interesting for several reasons: transducing a biological signal to an electric readout, using surface analytical tools such as Surface Plasmon Resonance (SPR, Infrared Reflection Absorption Spectroscopy, Neutron Reflectivity or Electrochemistry. The most widely used method to prepare supported lipid membranes is fusion of preexisting liposomes. It is quite efficient on hydrophilic surfaces such as glass, mica or SiO2, but vesicle fusion on metals and metal oxide surfaces (as gold, titanium oxide or indium tin oxide, remains a challenge, particularly for vesicles containing charged lipids, as is the case of bacterial lipids. We describe a simple method based on modifying the gold surface with a charged mercaptopropionic acid self-assembled monolayer and liposomes partially solubilized with detergent. The formed bilayers were characterized using a Quartz Crystal Microbalance with dissipation (QCM-D and Atomic Force Microscopy (AFM. Some advantages of this protocol are that the stability of the self-assembled monolayer allows for repeated use of the substrate after detergent removal of the bilayer and that the amount of detergent required for optimal fusion can be determined previously using the lipid-detergent solubility curve.
Pair interaction of bilayer-coated nanoscopic particles

International Nuclear Information System (INIS)

Qi-Yi, Zhang

2009-01-01

The pair interaction between bilayer membrane-coated nanosized particles has been explored by using the self-consistent field (SCF) theory. The bilayer membranes are composed of amphiphilic polymers. For different system parameters, the pair-interaction free energies are obtained. Particular emphasis is placed on the analysis of a sequence of structural transformations of bilayers on spherical particles, which occur during their approaching processes. For different head fractions of amphiphiles, the asymmetrical morphologies between bilayers on two particles and the inverted micellar intermediates have been found in the membrane fusion pathway. These results can benefit the fabrication of vesicles as encapsulation vectors for drug and gene delivery. (condensed matter: structure, thermal and mechanical properties)
Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function

Directory of Open Access Journals (Sweden)

Jakob Andersson

2016-05-01

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

Energy Technology Data Exchange (ETDEWEB)

Yun, Minhyuk; Lee, Eunho; Cho, Kilwon; Jeon, Sangmin, E-mail: jeons@postech.ac.kr [Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang (Korea, Republic of)

2014-08-18

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

International Nuclear Information System (INIS)

Northcutt, Robert; Sundaresan, Vishnu-Baba

2012-01-01

Conducting polymers are electroactive materials that undergo conformal relaxation of the polymer backbone in the presence of an electrical field through ion exchange with solid or aqueous electrolytes. This conformal relaxation and the associated morphological changes make conducting polymers highly suitable for actuation and sensing applications. Among smart materials, bioderived active materials also use ion transport for sensing and actuation functions via selective ion transport. The transporter proteins extracted from biological cell membranes and reconstituted into a bilayer lipid membrane in bioderived active materials regulate ion transport for engineering functions. The protein transporter reconstituted in the bilayer lipid membrane is referred to as the bioderived membrane and serves as the active component in bioderived active materials. Inspired by the similarities in the physics of transduction in conducting polymers and bioderived active materials, an integrated ionic device is formed from the bioderived membrane and the conducting polymer membrane. This ionic device is fabricated into a laminated thin-film membrane and a common ion that can be processed by the bioderived and the conducting polymer membranes couple the ionic function of these two membranes. An integrated ionic device, fabricated from polypyrrole (PPy) doped with sodium dodecylbenzenesulfonate (NaDBS) and an alamethicin-reconstituted DPhPC bilayer lipid membrane, is presented in this paper. A voltage-gated sodium current regulates the electrochemical response in the PPy(DBS) layer. The integrated device is fabricated on silicon-based substrates through microfabrication, electropolymerization, and vesicle fusion, and ionic activity is characterized through electrochemical measurements. (paper)
Lipid Bilayer Formation on Organic Electronic Materials

KAUST Repository

Zhang, Yi

2018-04-23

The lipid bilayer is the elemental structure of cell membrane, forming a stable barrier between the interior and exterior of the cell while hosting membrane proteins that enable selective transport of biologically important compounds and cellular recognition. Monitoring the quality and function of lipid bilayers is thus essential and can be performed using electrically active substrates that allow for transduction of signals. Such a promising electronic transducer material is the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) which has provided a plethora of novel bio transducing architectures. The challenge is however in assembling a bilayer on the conducting polymer surface, which is defect-free and has high mobility. Herein, we investigate the fusion of zwitterionic vesicles on a variety of PEDOT:PSS films, but also on an electron transporting, negatively charged organic semiconductor, in order to understand the surface properties that trigger vesicle fusion. The PEDOT:PSS films are prepared from dispersions containing different concentrations of ethylene glycol included as a formulation additive, which gives a handle to modulate surface physicochemical properties without a compromise on the chemical composition. The strong correlation between the polarity of the surface, the fusion of vesicles and the mobility of the resulting bilayer aides extracting design principles for the development of future conducting polymers that will enable the formation of lipid bilayers.
Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

Science.gov (United States)

Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

2016-03-29

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions
Gas Separation through Bilayer Silica, the Thinnest Possible Silica Membrane.

Science.gov (United States)

Yao, Bowen; Mandrà, Salvatore; Curry, John O; Shaikhutdinov, Shamil; Freund, Hans-Joachim; Schrier, Joshua

2017-12-13

Membrane-based gas separation processes can address key challenges in energy and environment, but for many applications the permeance and selectivity of bulk membranes is insufficient for economical use. Theory and experiment indicate that permeance and selectivity can be increased by using two-dimensional materials with subnanometer pores as membranes. Motivated by experiments showing selective permeation of H 2 /CO mixtures through amorphous silica bilayers, here we perform a theoretical study of gas separation through silica bilayers. Using density functional theory calculations, we obtain geometries of crystalline free-standing silica bilayers (comprised of six-membered rings), as well as the seven-, eight-, and nine-membered rings that are observed in glassy silica bilayers, which arise due to Stone-Wales defects and vacancies. We then compute the potential energy barriers for gas passage through these various pore types for He, Ne, Ar, Kr, H 2 , N 2 , CO, and CO 2 gases, and use the data to assess their capability for selective gas separation. Our calculations indicate that crystalline bilayer silica, which is less than a nanometer thick, can be a high-selectivity and high-permeance membrane material for 3 He/ 4 He, He/natural gas, and H 2 /CO separations.
Mechanical properties of electrospun bilayer fibrous membranes as potential scaffolds for tissue engineering.

Science.gov (United States)

Pu, Juan; Komvopoulos, Kyriakos

2014-06-01

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

DEFF Research Database (Denmark)

Pszon-Bartosz, Kamila Justyna; Hansen, Jesper S.; Stibius, Karin B.

2011-01-01

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We...... establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein...... reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 105 FomA proteins could be incorporated...
Exploiting lipopolysaccharide-induced deformation of lipid bilayers to modify membrane composition and generate two-dimensional geometric membrane array patterns

Energy Technology Data Exchange (ETDEWEB)

Adams, Peter G. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Swingle, Kirstie L. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Univ. of New Mexico, Albuquerque, NM (United States); Paxton, Walter F. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Nogan, John J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Stromberg, Loreen R. [Univ. of New Mexico, Albuquerque, NM (United States); Firestone, Millicent A. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Mukundan, Harshini [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); New Mexico Consortium, Los Alamos, NM (United States); Montaño, Gabriel A. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-05-27

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

Science.gov (United States)

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.
Regulation of membrane protein function by lipid bilayer elasticity-a single molecule technology to measure the bilayer properties experienced by an embedded protein

International Nuclear Information System (INIS)

Lundbaek, Jens August

2006-01-01

Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes, in the general regulation of membrane protein function, is unclear. This is to a large extent due to lack of a generally accepted framework in which to understand the many observations. The present review summarizes studies which have demonstrated that the hydrophobic interactions between a membrane protein and the host lipid bilayer provide an energetic coupling, whereby protein function can be regulated by the bilayer elasticity. The feasibility of this 'hydrophobic coupling mechanism' has been demonstrated using the gramicidin channel, a model membrane protein, in planar lipid bilayers. Using voltage-dependent sodium channels, N-type calcium channels and GABA A receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established
CONDENSED MATTER: STRUCTURE, THERMAL AND MECHANICAL PROPERTIES: Pair interaction of bilayer-coated nanoscopic particles

Science.gov (United States)

Zhang, Qi-Yi

2009-02-01

The pair interaction between bilayer membrane-coated nanosized particles has been explored by using the self-consistent field (SCF) theory. The bilayer membranes are composed of amphiphilic polymers. For different system parameters, the pair-interaction free energies are obtained. Particular emphasis is placed on the analysis of a sequence of structural transformations of bilayers on spherical particles, which occur during their approaching processes. For different head fractions of amphiphiles, the asymmetrical morphologies between bilayers on two particles and the inverted micellar intermediates have been found in the membrane fusion pathway. These results can benefit the fabrication of vesicles as encapsulation vectors for drug and gene delivery.

Structure and organization of nanosized-inclusion-containing bilayer membranes

Science.gov (United States)

Ren, Chun-Lai; Ma, Yu-Qiang

2009-07-01

Based on a considerable amount of experimental evidence for lateral organization of lipid membranes which share astonishingly similar features in the presence of different inclusions, we use a hybrid self-consistent field theory (SCFT)/density-functional theory (DFT) approach to deal with bilayer membranes embedded by nanosized inclusions and explain experimental findings. Here, the hydrophobic inclusions are simple models of hydrophobic drugs or other nanoparticles for biomedical applications. It is found that lipid/inclusion-rich domains are formed at moderate inclusion concentrations and disappear with the increase in the concentration of inclusions. At high inclusion content, chaining of inclusions occurs due to the effective depletion attraction between inclusions mediated by lipids. Meanwhile, the increase in the concentration of inclusions can also cause thickening of the membrane and the distribution of inclusions undergoes a layering transition from one-layer structure located in the bilayer midplane to two-layer structure arranged into the two leaflets of a bilayer. Our theoretical predictions address the complex interactions between membranes and inclusions suggesting a unifying mechanism which reflects the competition between the conformational entropy of lipids favoring the formation of lipid- and inclusion-rich domains in lipids and the steric repulsion of inclusions leading to the uniform dispersion.
Porous Materials to Support Bilayer Lipid Membranes for Ion Channel Biosensors

Directory of Open Access Journals (Sweden)

Thai Phung

2011-01-01

Full Text Available To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy. Bilayers supported by hydrophobic materials (PTFE, polycarbonate, nylon, and silanised silver had optimal resistance (14–19 GΩ and capacitance (0.8–1.6 μF values whereas those with low hydrophobicity did not form BLMs (PVDF or were short-lived (unsilanised silver. The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE-supported bilayers. Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method.
Filament networks attached to membranes: cytoskeletal pressure and local bilayer deformation

International Nuclear Information System (INIS)

Auth, Thorsten; Safran, S A; Gov, Nir S

2007-01-01

Several cell types, among them red blood cells, have a cortical, two-dimensional (2D) network of filaments sparsely attached to their lipid bilayer. In many mammalian cells, this 2D polymer network is connected to an underlying 3D, more rigid cytoskeleton. In this paper, we consider the pressure exerted by the thermally fluctuating, cortical network of filaments on the bilayer and predict the bilayer deformations that are induced by this pressure. We treat the filaments as flexible polymers and calculate the pressure that a network of such linear chains exerts on the bilayer; we then minimize the bilayer shape in order to predict the resulting local deformations. We compare our predictions with membrane deformations observed in electron micrographs of red blood cells. The polymer pressure along with the resulting membrane deformation can lead to compartmentalization, regulate in-plane diffusion and may influence protein sorting as well as transmit signals to the polymerization of the underlying 3D cytoskeleton
Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins
Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

Science.gov (United States)

Richards, Mark J; Daniel, Susan

2017-02-07

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

Science.gov (United States)

Morigaki, Kenichi; Mizutani, Kazuyuki; Saito, Makoto; Okazaki, Takashi; Nakajima, Yoshihiro; Tatsu, Yoshiro; Imaishi, Hiromasa

2013-02-26

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.
An Investigation on bilayer structures of electrospun polyacrylonitrile nanofibrous membrane and cellulose membrane used as filtration media for apple juice clarification

Science.gov (United States)

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

2018-05-01

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

NARCIS (Netherlands)

Moshtari Khah, S.; Oppers, N.A.W.; de Groot, M.T.; Keurentjes, J.T.F.; Schouten, J.C.; van der Schaaf, J.

2017-01-01

This work describes a model for bilayer cation-exchange membranes used in the chlor-alkali process. The ion transport inside the membrane is modeled with the Nernst–Planck equation. A logistic function is used at the boundary between the two layers of the bilayer membrane to describe the change in
Fluorescent molecular probes based on excited state prototropism in lipid bilayer membrane

Science.gov (United States)

Mohapatra, Monalisa; Mishra, Ashok K.

2012-03-01

Excited state prototropism (ESPT) is observed in molecules having one or more ionizable protons, whose proton transfer efficiency is different in ground and excited states. The interaction of various ESPT molecules like naphthols and intramolecular ESPT (ESIPT) molecules like hydroxyflavones etc. with different microheterogeneous media have been studied in detail and excited state prototropism as a probe concept has been gaining ground. The fluorescence of different prototropic forms of such molecules, on partitioning to an organized medium like lipid bilayer membrane, often show sensitive response to the local environment with respect to the local structure, physical properties and dynamics. Our recent work using 1-naphthol as an ESPT fluorescent molecular probe has shown that the incorporation of monomeric bile salt molecules into lipid bilayer membranes composed from dipalmitoylphosphatidylcholine (DPPC, a lung surfactant) and dimyristoylphosphatidylcholine (DMPC), in solid gel and liquid crystalline phases, induce appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (fisetin, an ESIPT molecule having antioxidant properties, in lipid bilayer membrane has been sensitively monitored from its intrinsic fluorescence behaviour.
Electric field-induced reorganization of two-component supported bilayer membranes.

Science.gov (United States)

Groves, J T; Boxer, S G; McConnell, H M

1997-12-09

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.
Membrane-on-a-Chip : Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion

NARCIS (Netherlands)

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

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with
Single-particle fusion of influenza viruses reveals complex interactions with target membranes

Science.gov (United States)

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

2018-05-01

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

Science.gov (United States)

Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

2018-02-01

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.
Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes

Energy Technology Data Exchange (ETDEWEB)

Camley, Brian A. [Center for Theoretical Biological Physics and Department of Physics, University of California, San Diego, California 92093 (United States); Department of Physics, University of California, Santa Barbara, California 93106 (United States); Lerner, Michael G. [Department of Physics and Astronomy, Earlham College, Richmond, Indiana 47374 (United States); Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 (United States); Pastor, Richard W. [Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 (United States); Brown, Frank L. H. [Department of Physics, University of California, Santa Barbara, California 93106 (United States); Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106 (United States)

2015-12-28

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

International Nuclear Information System (INIS)

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

2015-01-01

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

Energy Technology Data Exchange (ETDEWEB)

Huebner, J S; Arrieta, R T [University of North Florida, Jacksonville (USA); Naval Medical Research Inst., Bethesda, MD (USA))

1982-04-01

Ultraviolet light flashes induced voltage transients across bilayer lipid membranes when aromatic amino acids were adsorbed to one side of the membrane. These photo-effects varied with the chromophore structure, the aqueous solution salt concentration, pH and oxygen partial pressure. These photo-effects are attributed to the migration of electrically charged photochemical intermediates in the membrane, and provide a new method for studying the effects of UV light on membranes.
Mechanics of Lipid Bilayer Membranes

Science.gov (United States)

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.
Low frequency acoustic properties of bilayer membrane acoustic metamaterial with magnetic oscillator

Directory of Open Access Journals (Sweden)

Nansha Gao

2017-07-01

Full Text Available A bilayer membrane acoustic metamaterial was proposed to overcome the influence of the mass law on traditional acoustic materials and obtain a lightweight thin-layer structure that can effectively isolate low frequency noise. The finite element analysis (FEA results agree well with the experimental results. It is proved that the sound transmission losses (STLs of the proposed structures are higher than those of same surface density acoustic materials. The introduction of the magnetic mass block is different from the traditional design method, in which only a passive mass block is fixed on the membrane. The magnetic force will cause tension in the membrane, increase membrane prestress, and improve overall structural stiffness. The effects of the geometry size on the STLs are discussed in detail. The kind of method presented in this paper can provide a new means for engineering noise control. Keywords: Bilayer membrane acoustic metamaterial, Low frequency sound insulation, Sound transmission loss, Magnet oscillator
Semiconductor particle mediated photoelectron transfers in bilayer lipid membranes

International Nuclear Information System (INIS)

Fendler, J.H.; Baral, S.

1989-01-01

This paper discusses semiconductor particles in situ generated on the cis surface of glyceryl monooleate (GMO) bilayer lipid membranes (BLMs), that have been used to mediate photoelectric effects. The presence of semiconductors on the BLM surface is addressed. The observed photoelectric effects are rationalized and presented
Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy.

Science.gov (United States)

Khan, Muhammad Shuja; Dosoky, Noura Sayed; Patel, Darayas; Weimer, Jeffrey; Williams, John Dalton

2017-07-05

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature ( Tm ) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above-as well as below-the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10 -2 -10⁵ Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm . The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures.

Regulation of membrane protein function by lipid bilayer elasticity—a single molecule technology to measure the bilayer properties experienced by an embedded protein

DEFF Research Database (Denmark)

Lundbæk, Jens August

2008-01-01

, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes......-dependent sodium channels, N-type calcium channels and GABAA receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic...... properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established....
Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes

DEFF Research Database (Denmark)

Lundbæk, Jens August; Collingwood, S.A.; Ingolfsson, H.I.

2010-01-01

with collective physical properties (e.g. thickness, intrinsic monolayer curvature or elastic moduli). Studies in physico-chemical model systems have demonstrated that changes in bilayer physical properties can regulate membrane protein function by altering the energetic cost of the bilayer deformation associated...... with a protein conformational change. This type of regulation is well characterized, and its mechanistic elucidation is an interdisciplinary field bordering on physics, chemistry and biology. Changes in lipid composition that alter bilayer physical properties (including cholesterol, polyunsaturated fatty acids...... channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli....
Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

Science.gov (United States)

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.
Spatial orientation and electric-field-driven transport of hypericin inside of bilayer lipid membranes.

Science.gov (United States)

Strejčková, Alena; Staničová, Jana; Jancura, Daniel; Miškovský, Pavol; Bánó, Gregor

2013-02-07

Fluorescence experiments were carried out to investigate the interaction of hypericin (Hyp), a natural hydrophobic photosensitizer, with artificial bilayer lipid membranes. The spatial orientation of Hyp monomers incorporated in diphytanoyl phosphatidylcholine (DPhPC) membranes was determined by measuring the dependence of the Hyp fluorescence intensity on the angle of incidence of p- and s-polarized excitation laser beams. Inside of the membrane, Hyp monomers are preferentially located in the layers near the membrane/water interface and are oriented with the S(1) ← S(0) transition dipole moments perpendicular to the membrane surface. Transport of Hyp anions between the two opposite sides of the lipid bilayer was induced by applying rectangular electric field pulses to the membrane. The characteristic time for Hyp transport through the membrane center was evaluated by the analysis of the Hyp fluorescence signal during the voltage pulses. In the zero-voltage limit, the transport time approached 70 ms and gradually decreased with higher voltage applied to the membrane. In addition, our measurements indicated an apparent pK(a) constant of 8 for Hyp deprotonation in the membrane.
Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

International Nuclear Information System (INIS)

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

Directory of Open Access Journals (Sweden)

Heidi Koldsø

2014-10-01

Full Text Available 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.
Study of the ion-channel behavior on glassy carbon electrode supported bilayer lipid membranes stimulated by perchlorate anion

Energy Technology Data Exchange (ETDEWEB)

Zhang, Zhiquan; Shi, Jun; Huang, Weimin, E-mail: huangwm@jlu.edu.cn

2015-10-01

In this paper, a kind of didodecyldimethylammonium bromide (DDAB) layer membranes was supported on a glassy carbon electrode (GCE). We studied the ion channel behavior of the supported bilayer lipid membrane by scanning electrochemical microscopy (SCEM) in tris(2,2′-bipyridine) ruthenium(II) solution. Perchlorate anion was used as a presence of stimulus and ruthenium(II) complex cations as the probing ions for the measurement of SECM, the lipid membrane channel was opened and exhibited the behavior of distinct SECM positive feedback curve. The channel was in a closed state in the absence of perchlorate anions while reflected the behavior of SECM negative feedback curve. The rates of electron transfer reaction in the lipid membranes surface were detected and it was dependant on the potential of SECM. - Highlights: • The rates of electron transfer reaction in the lipid membranes surface were detected. • Dynamic investigations of ion-channel behavior of supported bilayer lipid membranes by scanning electrochemical microscopy • A novel way to explore the interaction between molecules and supported bilayer lipid membranes.
Bilayer porous scaffold based on poly-({epsilon}-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Zhou Zhihua; Zhou Yang [Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Chen Yiwang, E-mail: ywchen@ncu.edu.cn [Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Nie Huarong, E-mail: niehr@iccas.ac.cn [Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Wang Yang [First Affiliated Hospital, Nanchang University, 17 Yongwaizheng Road, Nanchang 330006 (China); Li Fan; Zheng Yan [Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China)

2011-12-15

Electrospun poly-({epsilon}-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0 Degree-Sign , the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66-77.3 MPa and 62.3-75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.
Bilayer porous scaffold based on poly-(ε-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

International Nuclear Information System (INIS)

Zhou Zhihua; Zhou Yang; Chen Yiwang; Nie Huarong; Wang Yang; Li Fan; Zheng Yan

2011-01-01

Electrospun poly-(ε-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0°, the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66-77.3 MPa and 62.3-75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.
Penetration and fusion of phospholipid vesicles by lysozyme

International Nuclear Information System (INIS)

Kim, J.; Kim, H.

1989-01-01

The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([ 125 I]iodophenyl)diazirine ([ 125 I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [ 125 I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion
Penetration and fusion of phospholipid vesicles by lysozyme

Energy Technology Data Exchange (ETDEWEB)

Kim, J.; Kim, H. (Korea Advanced Institute of Science and Technology, Seoul)

1989-10-01

The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-(({sup 125}I)iodophenyl)diazirine (({sup 125}I)TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent ({sup 125}I)TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.
Lipid Acrobatics in the Membrane Fusion Arena

NARCIS (Netherlands)

Markvoort, Albert J.; Marrink, Siewert J.; Chernomordik, Leonid V.; Kozlov, Michael M.

2011-01-01

In this review, we describe the recent contribution of computer simulation approaches to unravel the molecular details of membrane fusion. Over the past decade, fusion between apposed membranes and vesicles has been studied using a large variety of simulation methods and systems. Despite the variety
The Multifaceted Role of SNARE Proteins in Membrane Fusion.

Science.gov (United States)

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.
Statistical thermodynamics of association colloids : the equilibrium structure of micelles, vesicles, and bilayer membranes

NARCIS (Netherlands)

Leermakers, F.A.M.

1988-01-01

The aim of the present study was to unravel the general equilibrium physical properties of lipid bilayer membranes. We consider four major questions:
1. What determines the morphology of the association colloids (micelles, membranes, vesicles) in general?
2. Do the
Characterization of a structural intermediate of flavivirus membrane fusion.

Directory of Open Access Journals (Sweden)

Karin Stiasny

2007-02-01

Full Text Available Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion.
Study of pH (low) insertion peptides (pHLIPs) interaction with lipid bilayer of membrane

Science.gov (United States)

Weerakkody, Dhammika

The pH-dependent interactions of pHLIPsRTM (pH (Low) Insertion Peptides) with lipid bilayer of membrane provides an opportunity to study and address fundamental questions of protein folding/insertion into membrane and unfolding/exit, as well as develop novel approach to target acidic diseased tissue such as cancer, ischemic myocardium, infection and others. The main goal of the work presented here is to answer the following questions: - What is the molecular mechanism of spontaneous insertion and folding of a peptide in a lipid bilayer of membrane; - What is the molecular mechanism of unfolding and exit of a peptide from a lipid bilayer of membrane; - How polar cargo attached to a peptide's inserting end might affect the process of insertion into a lipid bilayer of membrane; How sequence variation will affect a peptide's interactions with a lipid bilayer of membrane (partitioning into bilayer at neutral and low pH; apparent pK of insertion) with the main goal to identify the best pHLIP variants for imaging and therapy of pathological states such as cancer and others. It has been demonstrated that pHLIP insertion into a membrane is associated with the protonation of Asp/Glu residues, which leads to an increase of hydrophobicity that triggers the folding and insertion of the peptide across a lipid bilayer. The insertion of the pHLIP is unidirectional and it is accompanied by the release of energy. Therefore, the energy of membrane associated-folding can be used to favor the movement of cell-impermeable polar cargo molecules across the hydrophobic membrane bilayer when they are attached to the inserting end of pHLIP. Both pH-targeting behavior and molecular translocation have been demonstrated in cultured cells and in vivo. Thus, there is an opportunity to develop a novel concept in drug delivery, which is based on the use of a monomeric, pH-sensitive peptide molecular transporter, to deliver agents that are significantly more polar than conventional drugs
Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

Energy Technology Data Exchange (ETDEWEB)

Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

2007-03-01

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.
Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

Directory of Open Access Journals (Sweden)

Doron Kabaso

2012-01-01

Full Text Available The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion or widen completely (full fusion to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.
Affinity of four polar neurotransmitters for lipid bilayer membranes

DEFF Research Database (Denmark)

Wang, Chunhua; Ye, Fengbin; Valardez, Gustavo F.

2011-01-01

. The simulations suggest that this attraction mainly relies on electrostatic interactions of the amino group of the neurotransmitter and the lipid phosphate. We conclude that moderate attraction to lipid membranes occurs for some polar neurotransmitters and hence that one premise for a theory of bilayer-mediated......Weak interactions of neurotransmitters and the lipid matrix in the synaptic membrane have been hypothesized to play a role in synaptic transmission of nerve signals, particularly with respect to receptor desensitization (Cantor, R. S. Biochemistry 2003, 42, 11891). The strength of such interactions......, however, was not measured, and this is an obvious impediment for further evaluation and understanding of a possible role for desensitization. We have used dialysis equilibrium to directly measure the net affinity of selected neurotransmitters for lipid membranes and analyzed this affinity data...
Post-Fusion Membrane Reorganization.

Science.gov (United States)

1993-01-27

diphosphoglycerate , and NEM (a crosslinking agent), and ethanol treatments all had reproducible and very specific effects on the kinetic phases and the fusion product...actually, at the ultrastructure level , a double membrane multiply perforated with fusion sites (or pores). Also, because the heat treatment was within...relationships. Moreover. 2.3- Diphosphoglycerate (2-3-DPG). a naturally occuring metabolite which is known to have a regulatory role in spectrin-cytoskeletal

Characterization of the functional requirements of West Nile virus membrane fusion.

Science.gov (United States)

Moesker, Bastiaan; Rodenhuis-Zybert, Izabela A; Meijerhof, Tjarko; Wilschut, Jan; Smit, Jolanda M

2010-02-01

Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the fluorophore, pyrene. Fusion of WNV with liposomes occurred on the timescale of seconds and was strictly dependent on mildly acidic pH. Optimal fusion kinetics were observed at pH 6.3, the threshold for fusion being pH 6.9. Preincubation of the virus alone at pH 6.3 resulted in a rapid loss of fusion capacity. WNV fusion activity is strongly promoted by the presence of cholesterol in the target membrane. Furthermore, we provide direct evidence that cleavage of prM to M is a requirement for fusion activity of WNV.
Fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity.

Science.gov (United States)

Lai, Alex L; Park, Heather; White, Judith M; Tamm, Lukas K

2006-03-03

The fusion peptide of influenza hemagglutinin is crucial for cell entry of this virus. Previous studies showed that this peptide adopts a boomerang-shaped structure in lipid model membranes at the pH of membrane fusion. To examine the role of the boomerang in fusion, we changed several residues proposed to stabilize the kink in this structure and measured fusion. Among these, mutants E11A and W14A expressed hemagglutinins with hemifusion and no fusion activities, and F9A and N12A had no effect on fusion, respectively. Binding enthalpies and free energies of mutant peptides to model membranes and their ability to perturb lipid bilayer structures correlated well with the fusion activities of the parent full-length molecules. The structure of W14A determined by NMR and site-directed spin labeling features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang of the wild-type, which points into the membrane. A specific fixed angle boomerang structure is thus required to support membrane fusion.
Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

International Nuclear Information System (INIS)

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

2016-01-01

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 10 8 –10 9 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 ⋅ 10 8 V m −1 ) when in the fluid phase with a monovalent counter-ion and ∼1.4 V (∼2.8 ⋅ 10 8 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
Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

Energy Technology Data Exchange (ETDEWEB)

Rønnest, A. K.; Peters, G. H.; Hansen, F. Y., E-mail: flemming@kemi.dtu.dk [Department of Chemistry, Technical University of Denmark, IK 207 DTU, DK-2800 Lyngby (Denmark); Taub, H.; Miskowiec, A. [Department of Physics and Astronomy and the University of Missouri Research Reactor,University of Missouri, Columbia, Missouri 65211 (United States)

2016-04-14

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 10{sup 8}–10{sup 9} V m{sup −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 ⋅ 10{sup 8} V m{sup −1}) when in the fluid phase with a monovalent counter-ion and ∼1.4 V (∼2.8 ⋅ 10{sup 8} V m{sup −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
Blocking of valinomycin-mediated bilayer membrane conductance by substituted benzimidazoles.

Science.gov (United States)

Kuo, K H; Fukuto, T R; Miller, T A; Bruner, L J

1976-01-01

Valinomycin selectively transports alkali cations, e.g. potassium ions, across lipid bilayer membranes. The blocking of this carrier-mediated transport by four substituted benzimidazoles has been investigated. The compounds are 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, (TTFB); 4,5,6,7,-tetrachloro-2-methylbenzimidazole, (TMB); 2-trifluoromethylbenzimidazole, (TFB); and 2-methylbenzimidazole, (MBM). Because of its low acidic dissociation constant (pKa = 5.04), the blocking efficiency of TTFB in both neutral and anionic forms in the aqueous phase could be studied. The compounds exhibit the blocking efficiency sequence, TTFB- greater than TTFB0 greater than TMB0 greater than TFB0 greater than MBM0. The corresponding scale of decreasing lipophilicity, as determined by octanol/water partitioning, is TTFB0 greater than TMB0 greater than TTFB- greater than TFB0 greater than MBM0. Comparison of neutral species establishes a positive correlation of blocking efficiency with lipophilicity, with the latter being conferred primarily by chlorination of the benzenoid nucleus. Anionic TTFB, on the other hand, is the most effective blocking agent studied in spite of the fact that its dissociation in the aqueous phase markedly impedes its entry (presumably as a neutral species) into a bulk hydrocarbon phase. This observation suggests that the blocking of valinomycin-mediated bilayer membrane conductance takes place at the membrane/solution interface. PMID:1247644
Quantitative optical microscopy and micromanipulation studies on the lipid bilayer membranes of giant unilamellar vesicles

DEFF Research Database (Denmark)

Bagatolli, Luis; Needham, David

2014-01-01

to study composition-structure-property materials relationships of free-standing lipid bilayer membranes. Because their size (~5 to 100 m diameter) that is well above the resolution limit of regular light microscopes, GUVs are suitable membrane models for optical microscopy and micromanipulation...
Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes

DEFF Research Database (Denmark)

Kopec, Wojciech; Telenius, Jelena; Khandelia, Himanshu

2013-01-01

Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many such molecu......Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many...
Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes.

Directory of Open Access Journals (Sweden)

Himanshu Khandelia

Full Text Available Triglycerides have a limited solubility, around 3%, in phosphatidylcholine lipid bilayers. Using millisecond-scale course grained molecular dynamics simulations, we show that the model lipid bilayer can accommodate a higher concentration of triolein (TO than earlier anticipated, by sequestering triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements.
Fusion of Sendai Virus with Vesicles of Oligomerizable Lipids: A Micro Calorimetric Analysis of Membrane Fusion

OpenAIRE

Ravoo, Bart Jan; Weringa, Wilke D.; Engberts, Jan B.F.N.

2000-01-01

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4-(beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37°C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sen...
Microchemical device based on microscopic bilayer lipid membranes; Bisho 2 bunshimaku wo mochiiita maikuro kagaku debaisu

Energy Technology Data Exchange (ETDEWEB)

Yokoyama, H. [Electrotechnical Lab., Ibaraki (Japan)

1996-04-01

If an organism is regarded as a macromolecular system, the element device to construct the same is the molecular structure of nano meter scale formed by the functional protein existing in biomembranes. A lot of essential functions of organism such as the sense reception including vision, gustation, etc., photosynthesis, energy-substance production and so on are performed therein. In this paper, the structure, preparing process and the functions of the microchemical device using micro-bilipid membranes are described. The simulation of the sense receiving functions of organisms is tried by said microchemical device wherein, same as biomembranes, the base is bilayer lipid molecular membrane and the receptive protein for receiving signals from exterior and output molecules such as ion channels connected to said receptive protein and the like are incorporated in the membranes. Recently, it becomes possible to make a partial imaging of the bilayer lipid membranes fixed on porous membrane by the observation with scanning Maxwell-stress microscope. 4 refs., 3 figs.
Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor

DEFF Research Database (Denmark)

Borch, Jonas; Torta, Federico; Sligar, Stephen G

2008-01-01

nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor G(M1) constitute...... a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs containing the glycolipid receptor G(M1) in lipid bilayers, enabling measurements of binding of its soluble interaction...
On the freezing behavior and diffusion of water in proximity to single-supported zwitterionic and anionic bilayer lipid membranes

DEFF Research Database (Denmark)

Miskowiec, A.; Buck, Z. N.; Brown, M. C.

2014-01-01

We compare the freezing/melting behavior of water hydrating single-supported bilayers of a zwitterionic lipid DMPC with that of an anionic lipid DMPG. For both membranes, the temperature dependence of the elastically scattered neutron intensity indicates distinct water types undergoing...... translational diffusion: bulk-like water probably located above the membrane and two types of confined water closer to the lipid head groups. The membranes differ in the greater width of the water freezing transition near the anionic DMPG bilayer compared to zwitterionic DMPC as well as in the abruptness...
Membrane Fusion Involved in Neurotransmission: Glimpse from Electron Microscope and Molecular Simulation

Directory of Open Access Journals (Sweden)

Zhiwei Yang

2017-06-01

Full Text Available Membrane fusion is one of the most fundamental physiological processes in eukaryotes for triggering the fusion of lipid and content, as well as the neurotransmission. However, the architecture features of neurotransmitter release machinery and interdependent mechanism of synaptic membrane fusion have not been extensively studied. This review article expounds the neuronal membrane fusion processes, discusses the fundamental steps in all fusion reactions (membrane aggregation, membrane association, lipid rearrangement and lipid and content mixing and the probable mechanism coupling to the delivery of neurotransmitters. Subsequently, this work summarizes the research on the fusion process in synaptic transmission, using electron microscopy (EM and molecular simulation approaches. Finally, we propose the future outlook for more exciting applications of membrane fusion involved in synaptic transmission, with the aid of stochastic optical reconstruction microscopy (STORM, cryo-EM (cryo-EM, and molecular simulations.
Membrane Fusion Involved in Neurotransmission: Glimpse from Electron Microscope and Molecular Simulation

Science.gov (United States)

Yang, Zhiwei; Gou, Lu; Chen, Shuyu; Li, Na; Zhang, Shengli; Zhang, Lei

2017-01-01

Membrane fusion is one of the most fundamental physiological processes in eukaryotes for triggering the fusion of lipid and content, as well as the neurotransmission. However, the architecture features of neurotransmitter release machinery and interdependent mechanism of synaptic membrane fusion have not been extensively studied. This review article expounds the neuronal membrane fusion processes, discusses the fundamental steps in all fusion reactions (membrane aggregation, membrane association, lipid rearrangement and lipid and content mixing) and the probable mechanism coupling to the delivery of neurotransmitters. Subsequently, this work summarizes the research on the fusion process in synaptic transmission, using electron microscopy (EM) and molecular simulation approaches. Finally, we propose the future outlook for more exciting applications of membrane fusion involved in synaptic transmission, with the aid of stochastic optical reconstruction microscopy (STORM), cryo-EM (cryo-EM), and molecular simulations. PMID:28638320
Model of SNARE-mediated membrane adhesion kinetics.

Directory of Open Access Journals (Sweden)

Jason M Warner

Full Text Available SNARE proteins are conserved components of the core fusion machinery driving diverse membrane adhesion and fusion processes in the cell. In many cases micron-sized membranes adhere over large areas before fusion. Reconstituted in vitro assays have helped isolate SNARE mechanisms in small membrane adhesion-fusion and are emerging as powerful tools to study large membrane systems by use of giant unilamellar vesicles (GUVs. Here we model SNARE-mediated adhesion kinetics in SNARE-reconstituted GUV-GUV or GUV-supported bilayer experiments. Adhesion involves many SNAREs whose complexation pulls apposing membranes into contact. The contact region is a tightly bound rapidly expanding patch whose growth velocity v(patch increases with SNARE density Gamma(snare. We find three patch expansion regimes: slow, intermediate, fast. Typical experiments belong to the fast regime where v(patch ~ (Gamma(snare(2/3 depends on SNARE diffusivities and complexation binding constant. The model predicts growth velocities ~10 - 300 microm/s. The patch may provide a close contact region where SNAREs can trigger fusion. Extending the model to a simple description of fusion, a broad distribution of fusion times is predicted. Increasing SNARE density accelerates fusion by boosting the patch growth velocity, thereby providing more complexes to participate in fusion. This quantifies the notion of SNAREs as dual adhesion-fusion agents.
Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Directory of Open Access Journals (Sweden)

A.T. Da Poian

2005-06-01

Full Text Available Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.
Transport Asymmetry of Novel Bi-Layer Hybrid Perfluorinated Membranes on the Base of MF-4SC Modified by Halloysite Nanotubes with Platinum

Directory of Open Access Journals (Sweden)

Anatoly Filippov

2018-03-01

Full Text Available Three types of bi-layer hybrid nanocomposites on the base of perfluorinated cation-exchange membrane MF-4SC (Russian analogue of Nafion®-117 were synthesized and characterized. It was found that two membranes possess the noticeable asymmetry of the current–voltage curve (CVC under changing their orientation towards the applied electric field, despite the absence of asymmetry of diffusion permeability. These phenomena were explained in the frame of the “fine-porous model” expanded for bi-layer membranes. A special procedure to calculate the real values of the diffusion layers thickness and the limiting current density was proposed. Due to asymmetry effects of the current voltage curves of bi-layer hybrid membranes on the base of MF-4SC, halloysite nanotubes and platinum nanoparticles, it is prospective to assemble membrane switches (membrane relays or diodes with predictable transport properties, founded upon the theory developed here.
Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

Science.gov (United States)

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.
The yeast cell fusion protein Prm1p requires covalent dimerization to promote membrane fusion.

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Alex Engel

2010-05-01

Full Text Available Prm1p is a multipass membrane protein that promotes plasma membrane fusion during yeast mating. The mechanism by which Prm1p and other putative regulators of developmentally controlled cell-cell fusion events facilitate membrane fusion has remained largely elusive. Here, we report that Prm1p forms covalently linked homodimers. Covalent Prm1p dimer formation occurs via intermolecular disulfide bonds of two cysteines, Cys-120 and Cys-545. PRM1 mutants in which these cysteines have been substituted are fusion defective. These PRM1 mutants are normally expressed, retain homotypic interaction and can traffic to the fusion zone. Because prm1-C120S and prm1-C545S mutants can form covalent dimers when coexpressed with wild-type PRM1, an intermolecular C120-C545 disulfide linkage is inferred. Cys-120 is adjacent to a highly conserved hydrophobic domain. Mutation of a charged residue within this hydrophobic domain abrogates formation of covalent dimers, trafficking to the fusion zone, and fusion-promoting activity. The importance of intermolecular disulfide bonding informs models regarding the mechanism of Prm1-mediated cell-cell fusion.
Impact of monoolein on aquaporin1-based supported lipid bilayer membranes

International Nuclear Information System (INIS)

Wang, Zhining; Wang, Xida; Ding, Wande; Wang, Miaoqi; Gao, Congjie; Qi, Xin

2015-01-01

Aquaporin (AQP) based biomimetic membranes have attracted considerable attention for their potential water purification applications. In this paper, AQP1 incorporated biomimetic membranes were prepared and characterized. The morphology and structure of the biomimetic membranes were characterized by in situ atomic force microscopy (AFM), infrared absorption spectroscopy, fluorescence microscopy, and contact angle measurements. The nanofiltration performance of the AQP1 incorporated membranes was investigated at 4 bar by using 2 g l −1 NaCl as feed solution. Lipid mobility plays an important role in the performance of the AQP1 incorporated supported lipid bilayer (SLB) membranes. We demonstrated that the lipid mobility is successfully tuned by the addition of monoolein (MO). Through in situ AFM and fluorescence recovery after photo-bleaching (FRAP) measurements, the membrane morphology and the molecular mobility were studied. The lipid mobility increased in the sequence DPPC < DPPC/MO (R MO = 5/5) < DOPC/MO (R MO = 5/5) < DOPC, which is consistent with the flux increment and salt rejection. This study may provide some useful insights for improving the water purification performance of biomimetic membranes. (paper)

Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

International Nuclear Information System (INIS)

Paredes, Angel M.; Ferreira, Davis; Horton, Michelle; Saad, Ali; Tsuruta, Hiro; Johnston, Robert; Klimstra, William; Ryman, Kate; Hernandez, Raquel; Chiu Wah; Brown, Dennis T.

2004-01-01

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a 'pore'-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell
Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

Directory of Open Access Journals (Sweden)

Daniel L Glauser

Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.
Membrane fusion and inverted phases

International Nuclear Information System (INIS)

Ellens, H.; Siegel, D.P.; Alford, D.; Yeagle, P.L.; Boni, L.; Lis, L.J.; Quinn, P.J.; Bentz, J.

1989-01-01

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31 P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31 P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates
Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study.

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Huai-Chun Chen

Full Text Available The second messenger lipid PIP(3 (phosphatidylinositol-3,4,5-trisphosphate is generated by the lipid kinase PI3K (phosphoinositide-3-kinase in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3-specific pleckstrin homology (PH domains to the membrane surface. Despite the broad importance of PIP(3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i PIP(3 target lipid that provides specificity and affinity, and (ii PS facilitator lipid that enhances the PIP(3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral
Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

International Nuclear Information System (INIS)

Lamb, Robert A.; Paterson, Reay G.; Jardetzky, Theodore S.

2006-01-01

Paramyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process-an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1-5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described
The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

NARCIS (Netherlands)

Bosch, B.J.

2004-01-01

The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion
Fusion of biological membranes

Indian Academy of Sciences (India)

Home; Journals; Pramana – Journal of Physics; Volume 64; Issue 6. Fusion of biological membranes. K Katsov M Müller M Schick. Invited Talks:- Topic 11. Biologically motivated problems (protein-folding models, dynamics at the scale of the cell; biological networks, evolution models, etc.) Volume 64 Issue 6 June 2005 pp ...
The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

International Nuclear Information System (INIS)

Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

2010-01-01

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.
Temperature dependence of positronium reactivities with charge transfer molecules in bilayer membranes

International Nuclear Information System (INIS)

Jean, Y.C.; Yu, C.; Wang, Y.Y.; Yeh, Y.Y.

1984-01-01

Rate constants for positronium atoms reacting chemically with charge-transfer molecules such as p-benzoquinone, nitrobenzene, and coenzyme Q-10 in a model bilayer membrane, dipalmitoylphosphatidylcholine (DPPC), have been measured at temperatures between 23 and 65 0 C. A strong variation of the positronium chemical reactivities, k/sub Ps/ was observed in these systems: k/sub Ps/ increases with increasing temperature until the pretransition temperature of the membrane reaches a maximum value near the main transition temperature and decreases at temperatures higher than the main transition temperature. This variation is interpreted in terms of fluidity and permeability changes associated with the phase transitions of membranes and in terms of charge-transfer-complex formation between the solubilized molecules and the polar head of the membrane. These results demonstrate that positronium and its annihilation characteristics can be employed to investigate charge transport phenomena and microstructural changes of real biological membranes
Cholesterol Protects the Oxidized Lipid Bilayer from Water Injury

DEFF Research Database (Denmark)

Owen, Michael C; Kulig, Waldemar; Rog, Tomasz

2018-01-01

In an effort to delineate how cholesterol protects membrane structure under oxidative stress conditions, we monitored the changes to the structure of lipid bilayers comprising 30 mol% cholesterol and an increasing concentration of Class B oxidized 1-palmitoyl-2-oleoylphosphatidylcholine (POPC...... in a characteristic reduction in bilayer thickness and increase in area per lipid, thereby increasing the exposure of the membrane hydrophobic region to water. However, cholesterol was observed to help reduce water injury by moving into the bilayer core and forming more hydrogen bonds with the oxPLs. Cholesterol also...... resists altering its tilt angle, helping to maintain membrane integrity. Water that enters the 1-nm-thick core region remains part of the bulk water on either side of the bilayer, with relatively few water molecules able to traverse through the bilayer. In cholesterol-rich membranes, the bilayer does...
MEMBRANE-FUSION OF SEMLIKI FOREST VIRUS INVOLVES HOMOTRIMERS OF THE FUSION PROTEIN

NARCIS (Netherlands)

WAHLBERG, JM; WILSCHUT, J; GAROFF, H

1992-01-01

Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion Processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated
Effects of carotenoids on lipid bilayers.

Science.gov (United States)

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

2018-01-31

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

Science.gov (United States)

Tamm, Lukas K

2003-07-11

Substantial progress has been made in recent years to augment the current understanding of structures and interactions that promote viral membrane fusion. This progress is reviewed with a particular emphasis on recently determined structures of viral fusion domains and their interactions with lipid membranes. The results from the different structural and thermodynamic experimental approaches are synthesized into a new proposed mechanism, termed the "spring-loaded boomerang" mechanism of membrane fusion, which is presented here as a hypothesis.
Effect of vesicle size on the prodan fluorescence in diheptadecanoylphosphatidylcholine bilayer membrane under atmospheric and high pressures.

Science.gov (United States)

Goto, Masaki; Sawaguchi, Hiroshi; Tamai, Nobutake; Matsuki, Hitoshi; Kaneshina, Shoji

2010-08-17

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they
DSC and EPR investigations on effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within lipid bilayer membrane.

Science.gov (United States)

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

2007-06-29

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

Science.gov (United States)

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

2017-10-27

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

Energy Technology Data Exchange (ETDEWEB)

Hattori, Masashi [Institute for Materials Chemistry and Engineering, Kyushu University, Kasuga 816-8580 (Japan); Noda, Kei, E-mail: nodakei@elec.keio.ac.jp [Department of Electronics and Electrical Engineering, Keio University, Hiyoshi, Yokohama 223-8522 (Japan)

2015-12-01

Graphical abstract: - Highlights: • A bilayer membrane composed of TiO{sub 2} nanotube array and palladium was fabricated. • The TiO{sub 2}/Pd bilayer membrane was prepared with an all-electrochemical process. • The membrane consists of pure Pd and anatase TiO{sub 2} nanotubes with no alloy formation. • Photocatalytic H{sub 2} production and concomitant separation were demonstrated. • High-purity H{sub 2} production rate and apparent quantum yield were evaluated. - Abstract: We developed an all-electrochemical technique for fabricating a bilayer structure of a titanium dioxide (TiO{sub 2}) nanotube array (TNA) and a palladium film (TNA/Pd membrane), which works for photocatalytic high-purity hydrogen production. Electroless plating was used for depositing the Pd film on the TNA surface prepared by anodizing a titanium foil. A 3-μm-thick TNA/Pd membrane without any pinholes in a 1.5-cm-diameter area was fabricated by transferring a 1-μm-thick TNA onto an electroless-plated 2-μm-thick Pd film with a mechanical peel-off process. This ultrathin membrane with sufficient mechanical robustness showed photocatalytic H{sub 2} production via methanol reforming under ultraviolet illumination on the TNA side, immediately followed by the purification of the generated H{sub 2} gas through the Pd layer. The hydrogen production rate and the apparent quantum yield for high-purity H{sub 2} production from methanol/water mixture with the TNA/Pd membrane were also examined. This work suggests that palladium electroless plating is more suitable and practical for preparing a well-organized TNA/Pd heterointerface than palladium sputter deposition.
Dispersion of fullerenes in phospholipid bilayers and the subsequent phase changes in the host bilayers

Energy Technology Data Exchange (ETDEWEB)

Jeng, U-S. [National Synchrotron Radiation Research Center, Hsinchu 30077, Taiwan (China)]. E-mail: usjeng@nsrrc.org.tw; Hsu, C.-H. [National Synchrotron Radiation Research Center, Hsinchu 30077, Taiwan (China); Lin, T.-L. [Department of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Wu, C.-M. [Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Chen, H.-L. [Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Tai, L.-A. [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Hwang, K.-C. [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China)

2005-02-28

We have studied the structure and phase transition characteristics of the fullerenes (C{sub 60})-embedded lipid bilayers. With small-angle neutron scattering (SANS), we have observed a degradation of bilayer ordering and a suppression effect on the phase transitions of the host vesicle bilayers of dipalmitoylphosphatidylcholine (DPPC), due to the embedment of fullerenes. The fullerene-embedded lipid system with substrate-oriented bilayers is also investigated using X-ray reflectivity and grazing incident small-angle X-ray scattering (GISAXS). In the depth direction, the multilamellar peaks observed in the X-ray reflectivity profile for the oriented DPPC/C{sub 60} bilayers reveal a larger head-to-head distance D{sub HH} of 50.6 A and a bilayer spacing D of 59.8 A, compared to the D{sub HH}=47.7 A and D=59.5 A for a pure DPPC membrane measured at the same conditions. Furthermore, the lipid head layers and water layers in the extracted electron density profile for the complex system are highly smeared, implying a fluctuating or corrugated structure in this zone. Correspondingly, GISAXS for the oriented DPPC/C{sub 60} membrane reveals stronger diffuse scatterings along the membrane plane than that for the pure DPPC system, indicating a higher in-plane correlation associated with the embedded fullerenes.
Decoupling of bilayer leaflets under gas supersaturation: nitrogen nanobubbles in a membrane and their implication in decompression sickness

Science.gov (United States)

Li, Jing; Zhang, Xianren; Cao, Dapeng

2018-05-01

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

Indian Academy of Sciences (India)

Home; Journals; Resonance – Journal of Science Education; Volume 19; Issue 5. Membrane Trafficking and Vesicle Fusion: Post-Palade Era Researchers Win the Nobel Prize. Riddhi Atul Jani Subba Rao Gangi Setty. General Article Volume 19 Issue 5 May 2014 pp 421-445 ...

A tethering complex drives the terminal stage of SNARE-dependent membrane fusion

Science.gov (United States)

D'Agostino, Massimo; Risselada, Herre Jelger; Lürick, Anna; Ungermann, Christian; Mayer, Andreas

2017-11-01

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore. This last step is highly energy-demanding. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.
Reactions of radicals with lecithin bilayers

International Nuclear Information System (INIS)

Barber, D.J.W.; Thomas, J.K.

1978-01-01

The kinetics of reaction of .OH and e/sub aq/ - with lecithin bilayers have been measured. The rate for .OH + lecithin is 5.1 +- 0.9 x 10 8 M -1 sec -1 while the e/sub aq/ - + lecithin rate is very slow. When a solute such as pyrene is solubilized in the bilayer, .OH and e/sub aq/ - may react with the solute; rates of 1.65 +- 0.12 x 10 9 M -1 sec -1 and 7 x 10 7 M -1 sec -1 have been measured for reaction of .OH and e/sub aq/ - , respectively, with pyrene in lecithin. These rates are lower than those observed for similar reactions in homogeneous systems. This is explained in terms of (a) the protective effect of the bilayer, this being especially true for e/sub aq/ - which does not readily leave the aqueous phase, and (b) in terms of the restricted diffusion imposed on the reactive species by the bilayer. The kinetics in these model systems are relevant to reactions of radicals with membranes. Long-term alteration in the model membrane following .OH attack is manifested in terms of damage to the head group, increasing water penetration of the bilayer, and of cross-linking with the membrane, thereby restricting motion in the interior of the bilayer. Increased rigidity and leakiness of membranes is an expected consequence of radiation damage
DNA nanotechnology: Bringing lipid bilayers into shape

Science.gov (United States)

Howorka, Stefan

2017-07-01

Lipid bilayers form the thin and floppy membranes that define the boundary of compartments such as cells. Now, a method to control the shape and size of bilayers using DNA nanoscaffolds has been developed. Such designer materials advance synthetic biology and could find use in membrane research.
Antimicrobial peptides at work: interaction of myxinidin and its mutant WMR with lipid bilayers mimicking the P. aeruginosa and E. coli membranes

Science.gov (United States)

Lombardi, Lucia; Stellato, Marco Ignazio; Oliva, Rosario; Falanga, Annarita; Galdiero, Massimiliano; Petraccone, Luigi; D'Errico, Geradino; de Santis, Augusta; Galdiero, Stefania; Del Vecchio, Pompea

2017-03-01

Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin.
Thermotropic and Barotropic Phase Behavior of Phosphatidylcholine Bilayers

Directory of Open Access Journals (Sweden)

Nobutake Tamai

2013-01-01

Full Text Available Bilayers formed by phospholipids are frequently used as model biological membranes in various life science studies. A characteristic feature of phospholipid bilayers is to undergo a structural change called a phase transition in response to environmental changes of their surroundings. In this review, we focus our attention on phase transitions of some major phospholipids contained in biological membranes, phosphatidylcholines (PCs, depending on temperature and pressure. Bilayers of dipalmitoylphosphatidylcholine (DPPC, which is the most representative lipid in model membrane studies, will first be explained. Then, the bilayer phase behavior of various kinds of PCs with different molecular structures is revealed from the temperature–pressure phase diagrams, and the difference in phase stability among these PC bilayers is discussed in connection with the molecular structure of the PC molecules. Furthermore, the solvent effect on the phase behavior is also described briefly.
Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity.

Science.gov (United States)

Kashiwada, Ayumi; Yamane, Iori; Tsuboi, Mana; Ando, Shun; Matsuda, Kiyomi

2012-01-31

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system. © 2011 American Chemical Society
Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

Science.gov (United States)

Ronzone, Erik; Paumet, Fabienne

2013-01-01

Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.
Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

Directory of Open Access Journals (Sweden)

Erik Ronzone

Full Text Available Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.
Simulation of polyethylene glycol and calcium-mediated membrane fusion

International Nuclear Information System (INIS)

Pannuzzo, Martina; De Jong, Djurre H.; Marrink, Siewert J.; Raudino, Antonio

2014-01-01

We report on the mechanism of membrane fusion mediated by polyethylene glycol (PEG) and Ca 2+ by means of a coarse-grained molecular dynamics simulation approach. Our data provide a detailed view on the role of cations and polymer in modulating the interaction between negatively charged apposed membranes. The PEG chains cause a reduction of the inter-lamellar distance and cause an increase in concentration of divalent cations. When thermally driven fluctuations bring the membranes at close contact, a switch from cis to trans Ca 2+ -lipid complexes stabilizes a focal contact acting as a nucleation site for further expansion of the adhesion region. Flipping of lipid tails induces subsequent stalk formation. Together, our results provide a molecular explanation for the synergistic effect of Ca 2+ and PEG on membrane fusion
A comparison of sulfur mustard and heptane penetrating a dipalmitoylphosphatidylcholine bilayer membrane

Energy Technology Data Exchange (ETDEWEB)

Mueller, Thomas J., E-mail: t.mueller@theo.chemie.tu-darmstadt.de [Theoretische Physikalische Chemie, Eduard-Zintl-Institut fuer Anorganische und Physikalische Chemie, Technische Universitaet Darmstadt, Petersenstrasse 20, 64287 Darmstadt (Germany); Mueller-Plathe, Florian [Theoretische Physikalische Chemie, Eduard-Zintl-Institut fuer Anorganische und Physikalische Chemie, Technische Universitaet Darmstadt, Petersenstrasse 20, 64287 Darmstadt (Germany)

2009-08-30

In the present molecular dynamics simulations we study the chemical warfare agent sulfur mustard (bis(2-chloroethyl) sulfide) and the alkane heptane inserted into a dipalmitoylphosphatidylcholine (DPPC) bilayer, a generic model for a biological membrane. We investigate the diffusion, the orientation, the preferred positioning, and the end-to-end distance of the solutes within the membrane as well as the corresponding coupling times. We compare results of equilibrium simulations and simulation at different external forces, which drag the solutes through the membrane. These properties lead to a general comparison of the rotational and translational behaviors of the two solutes during the penetration of the membrane. We show that sulfur mustard, due to its atomic charge polarization, its bigger flexibility and its smaller molecular volume, is the faster moving molecule within the membrane. In last consequence, we show that this leads to different limits for the transport mechanism as observed in these simulations. For heptane the hindrance to penetrate into the membrane is significantly higher than for sulfur mustard. In contrast to heptane molecules, which spend the most of the time penetrating the tail groups, sulfur mustard needs more time to escape the tail group-head group interface of the membrane.
Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

Science.gov (United States)

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.
Adsorption and Orientation of Human Islet Amyloid Polypeptide (hIAPP Monomer at Anionic Lipid Bilayers: Implications for Membrane-Mediated Aggregation

Directory of Open Access Journals (Sweden)

Guanghong Wei

2013-03-01

Full Text Available Protein misfolding and aggregation cause serious degenerative diseases, such as Alzheimer’s and type II diabetes. Human islet amyloid polypeptide (hIAPP is the major component of amyloid deposits found in the pancreas of type II diabetic patients. Increasing evidence suggests that β-cell death is related to the interaction of hIAPP with the cellular membrane, which accelerates peptide aggregation. In this study, as a first step towards understanding the membrane-mediated hIAPP aggregation, we investigate the atomic details of the initial step of hIAPP-membrane interaction, including the adsorption orientation and conformation of hIAPP monomer at an anionic POPG lipid bilayer by performing all-atom molecular dynamics simulations. We found that hIAPP monomer is quickly adsorbed to bilayer surface, and the adsorption is initiated from the N-terminal residues driven by strong electrostatic interactions of the positively-charged residues K1 and R11 with negatively-charged lipid headgroups. hIAPP binds parallel to the lipid bilayer surface as a stable helix through residues 7–22, consistent with previous experimental study. Remarkably, different simulations lead to the same binding orientation stabilized by electrostatic and H-bonding interactions, with residues R11, F15 and S19 oriented towards membrane and hydrophobic residues L12, A13, L16 and V17 exposed to solvent. Implications for membrane-mediated hIAPP aggregation are discussed.
Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

Science.gov (United States)

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.
Electrodeformation of multi-bilayer spherical concentric membranes by AC electric fields

Science.gov (United States)

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

2017-09-01

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

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Rao, Laxminarsimha V., E-mail: laxman@iitk.ac.in [Mechanics and Applied Mathematics Group, Department of Mechanical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Roy, Subhradeep [Department of Biomedical Engineering and Mechanics (MC 0219), Virginia Tech, 495 Old Turner Street, Blacksburg, VA 24061 (United States); Das, Sovan Lal [Mechanics and Applied Mathematics Group, Department of Mechanical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India)

2017-01-15

We estimate the equilibrium size distribution of cholesterol rich micro-domains on a lipid bilayer by solving Smoluchowski equation for coagulation and fragmentation. Towards this aim, we first derive the coagulation kernels based on the diffusion behaviour of domains moving in a two dimensional membrane sheet, as this represents the reality better. We incorporate three different diffusion scenarios of domain diffusion into our coagulation kernel. Subsequently, we investigate the influence of the parameters in our model on the coagulation and fragmentation behaviour. The observed behaviours of the coagulation and fragmentation kernels are also manifested in the equilibrium domain size distribution and its first moment. Finally, considering the liquid domains diffusing in a supported lipid bilayer, we fit the equilibrium domain size distribution to a benchmark solution.
Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature

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Philip P. Cheney

2017-03-01

Full Text Available 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.
Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

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Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.
Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

DEFF Research Database (Denmark)

Søe, Kent; Andersen, Thomas Lykke; Hobolt-Pedersen, Anne-Sofie

2011-01-01

fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer....... This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic...
Effect of intra-membrane C60 fullerenes on the modulus of elasticity and the mechanical resistance of gel and fluid lipid bilayers

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Zhou, Jihan; Liang, Dehai; Contera, Sonia

2015-10-01

Penetration and partition of C60 to the lipid bilayer core are both relevant to C60 toxicity, and useful to realise C60 biomedical potential. A key aspect is the effect of C60 on bilayer mechanical properties. Here, we present an experimental study on the mechanical effect of the incorporation of C60 into the hydrophobic core of fluid and gel phase zwitterionic phosphatidylcholine (PC) lipid bilayers. We demonstrate its incorporation inside the hydrophobic lipid core and the effect on the packing of the lipids and the vesicle size using a combination of infrared (IR) spectroscopy, atomic force microscopy (AFM) and laser light scattering. Using AFM we measured the Young's modulus of elasticity (E) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the absence (presence) of intra-membranous C60 at 24.5 °C. E of fluid phase supported bilayers is not altered by C60, but E increases with incorporation of C60 in gel phase bilayers. The increase is higher for longer hydrocarbon chains: 1.6 times for DPPC and 2 times for DSPC. However the mechanical resistance of gel phase bilayers of curved bilayered structures decreases with the incorporation of C60. Our combined results indicate that C60 causes a decrease in gel phase lipid mobility, i.e. an increase in membrane viscosity.
Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases

International Nuclear Information System (INIS)

Siegel, D.P.; Banschbach, J.; Alford, D.; Ellens, H.; Lis, L.J.; Quinn, P.J.; Yeagle, P.L.; Bentz, J.

1989-01-01

In a previous paper, it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31 P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, ΔT I , below the L α /H II phase transition temperature, T H . T H and ΔT I can be extremely sensitive to the lipid composition. The present work shows that 2 mol % of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower T H , ΔT I , and the temperature for fast membrane fusion by 15-20 degree C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo

Exploring the Local Elastic Properties of Bilayer Membranes Using Molecular Dynamics Simulations

DEFF Research Database (Denmark)

Pieffet, Gilles; Botero, Alonso; Peters, Günther H.J.

2014-01-01

Membrane mechanical elastic properties regulate a variety of cellular processes involving local membrane deformation, such as ion channel function and vesicle fusion. In this work, we used molecular dynamics simulations to estimate the local elastic properties of a membrane. For this, we calculated...... the stretching process in molecular detail, allowing us to fit this profile to a previously proposed continuum elastic model. Through this approach, we calculated an effective membrane spring constant of 42 kJ-2.mol-1, which is in good agreement with the PMF calculation. Furthermore, the solvation energy we...
Mitochondrial fusion through membrane automata.

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Giannakis, Konstantinos; Andronikos, Theodore

2015-01-01

Studies have shown that malfunctions in mitochondrial processes can be blamed for diseases. However, the mechanism behind these operations is yet not sufficiently clear. In this work we present a novel approach to describe a biomolecular model for mitochondrial fusion using notions from the membrane computing. We use a case study defined in BioAmbient calculus and we show how to translate it in terms of a P automata variant. We combine brane calculi with (mem)brane automata to produce a new scheme capable of describing simple, realistic models. We propose the further use of similar methods and the test of other biomolecular models with the same behaviour.
Single-molecule study of full-length NaChBac by planar lipid bilayer recording.

Directory of Open Access Journals (Sweden)

Andrew Jo

Full Text Available Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts. In this work, we investigated the single-molecule conductance and ionic selectivity behavior of NaChBac. Purified NaChBac protein was first reconstituted into lipid vesicles, which is subsequently incorporated into planar lipid bilayer by fusion. At single-molecule level, we were able to observe three distinct long-lived conductance sub-states of NaChBac. Change in the membrane potential switches on the channel mainly by increasing its opening probability. In addition, we found that individual NaChBac has similar permeability for Na+, K+, and Ca2+. The single-molecule behavior of the full-length protein is essentially highly stochastic. Our results show that planar lipid bilayer device can be used to study purified ion channels at single-molecule level in an artificial environment, and such studies can reveal new protein properties that are otherwise not observable in in vivo ensemble studies.
Structure and distribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes

International Nuclear Information System (INIS)

Puntheeranurak, Theeraporn; Stroh, Cordula; Zhu Rong; Angsuthanasombat, Chanan; Hinterdorfer, Peter

2005-01-01

Bacillus thuringiensis Cry δ-endotoxins cause death of susceptible insect larvae by forming lytic pores in the midgut epithelial cell membranes. The 65 kDa trypsin activated Cry4Ba toxin was previously shown to be capable of permeabilizing liposomes and forming ionic channels in receptor-free planar lipid bilayers. Here, magnetic ACmode (MACmode) atomic force microscopy (AFM) was used to characterize the lateral distribution and the native molecular structure of the Cry4Ba toxin in the membrane. Liposome fusion and the Langmuir-Blodgett technique were employed for supported lipid bilayer preparations. The toxin preferentially inserted in a self-assembled structure, rather than as a single monomeric molecule. In addition, the spontaneous insertion into receptor-free lipid bilayers lead to formation of characteristic pore-like structures with four-fold symmetry, suggesting that tetramers are the preferred oligomerization state of this toxin
A Neutron View of Proteins in Lipid Bilayers

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White, Stephen

2012-02-01

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly-charged S1-S4 voltage- sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated potassium channels. We have used neutron diffraction, solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field.
Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

2013-04-05

Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.
pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion.

Science.gov (United States)

Batishchev, O V; Shilova, L A; Kachala, M V; Tashkin, V Y; Sokolov, V S; Fedorova, N V; Baratova, L A; Knyazev, D G; Zimmerberg, J; Chizmadzhev, Y A

2016-01-01

Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In
Conflicting views on the membrane fusion machinery and the fusion pore

DEFF Research Database (Denmark)

Sørensen, Jakob B

2009-01-01

of the assembly of the fusogenic SNARE-complex. Here, I review conflicting views on the function of the core fusion machinery consisting of the SNAREs, Munc18, complexin, and synaptotagmin. Munc18 controls docking of vesicles to the plasma membrane and initial SNARE-complex assembly, whereas complexin...
Probing Lipid Bilayers under Ionic Imbalance.

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Lin, Jiaqi; Alexander-Katz, Alfredo

2016-12-06

Biological membranes are normally under a resting transmembrane potential (TMP), which originates from the ionic imbalance between extracellular fluids and cytosols, and serves as electric power storage for cells. In cell electroporation, the ionic imbalance builds up a high TMP, resulting in the poration of cell membranes. However, the relationship between ionic imbalance and TMP is not clearly understood, and little is known about the effect of ionic imbalance on the structure and dynamics of biological membranes. In this study, we used coarse-grained molecular dynamics to characterize a dipalmitoylphosphatidylcholine bilayer system under ionic imbalances ranging from 0 to ∼0.06 e charges per lipid (e/Lip). We found that the TMP displayed three distinct regimes: 1) a linear regime between 0 and 0.045 e/Lip, where the TMP increased linearly with ionic imbalance; 2) a yielding regime between ∼0.045 and 0.060 e/Lip, where the TMP displayed a plateau; and 3) a poration regime above ∼0.060 e/Lip, where we observed pore formation within the sampling time (80 ns). We found no structural changes in the linear regime, apart from a nonlinear increase in the area per lipid, whereas in the yielding regime the bilayer exhibited substantial thinning, leading to an excess of water and Na + within the bilayer, as well as significant misalignment of the lipid tails. In the poration regime, lipid molecules diffused slightly faster. We also found that the fluid-to-gel phase transition temperature of the bilayer dropped below the normal value with increased ionic imbalances. Our results show that a high ionic imbalance can substantially alter the essential properties of the bilayer, making the bilayer more fluid like, or conversely, depolarization of a cell could in principle lead to membrane stiffening. Copyright Â© 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
[3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

International Nuclear Information System (INIS)

Hamilton, S.L.; Alvarez, R.M.; Fill, M.; Hawkes, M.J.; Brush, K.L.; Schilling, W.P.; Stefani, E.

1989-01-01

Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [ 3 H]PN200-110, and the alkaloid, [ 3 H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling
Reconstitution of a Kv channel into lipid membranes for structural and functional studies.

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Lee, Sungsoo; Zheng, Hui; Shi, Liang; Jiang, Qiu-Xing

2013-07-13

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.
Shear-Induced Membrane Fusion in Viscous Solutions

KAUST Repository

Kogan, Maxim; Feng, Bobo; Nordé n, Bengt; Rocha, Sandra; Beke-Somfai, Tamá s

2014-01-01

Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We
The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

Directory of Open Access Journals (Sweden)

Chengliang Zhang

Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.
Insertion of Neurotransmitters into a Lipid Bilayer Membrane and Its Implication on Membrane Stability: A Molecular Dynamics Study.

Science.gov (United States)

Shen, Chun; Xue, Minmin; Qiu, Hu; Guo, Wanlin

2017-03-17

The signaling molecules in neurons, called neurotransmitters, play an essential role in the transportation of neural signals, during which the neurotransmitters interact with not only specific receptors, but also cytomembranes, such as synaptic vesicle membranes and postsynaptic membranes. Through extensive molecular dynamics simulations, the atomic-scale insertion dynamics of typical neurotransmitters, including methionine enkephalin (ME), leucine enkephalin (LE), dopamine (DA), acetylcholine (ACh), and aspartic acid (ASP), into lipid bilayers is investigated. The results show that the first three neurotransmitters (ME, LE, and DA) are able to diffuse freely into both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) membranes, and are guided by the aromatic residues Tyr and Phe. Only a limited number of these neurotransmitters are allowed to penetrate into the membrane, which suggests an intrinsic mechanism by which the membrane is protected from being destroyed by excessive inserted neurotransmitters. After spontaneous insertion, the neurotransmitters disturb the surrounding phospholipids in the membrane, as indicated by the altered distribution of components in lipid leaflets and the disordered lipid tails. In contrast, the last two neurotransmitters (ACh and ASP) cannot enter the membrane, but instead always diffuse freely in solution. These findings provide an understanding at the atomic level of how neurotransmitters interact with the surrounding cytomembrane, as well as their impact on membrane behavior. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers

International Nuclear Information System (INIS)

Mote, Kaustubh R.; Gopinath, T.; Traaseth, Nathaniel J.; Kitchen, Jason; Gor’kov, Peter L.; Brey, William W.; Veglia, Gianluigi

2011-01-01

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1 H- 15 N dipolar couplings (DC) and 15 N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([ 1 H, 15 N]-SE-PISEMA-PDSD). The incorporation of 2D 15 N/ 15 N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15 N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.
Low-pH-induced transformation of bilayer membrane into bicontinuous cubic phase in dioleoylphosphatidylserine/monoolein membranes.

Science.gov (United States)

Okamoto, Yoshihide; Masum, Shah Md; Miyazawa, Haruna; Yamazaki, Masahito

2008-04-01

Cubic biomembranes, nonbilayer membranes with connections in three-dimensional space that have a cubic symmetry, have been observed in various cells. Interconversion between the bilayer liquid-crystalline (L(alpha)) phase and cubic phases attracted much attention in terms of both biological and physicochemical aspects. Herein we report the pH effect on the phase and structure of dioleoylphosphatidylserine (DOPS)/monoolein (MO) membranes under a physiological ion concentration condition, which was revealed by small-angle X-ray scattering (SAXS) measurement. At neutral pH, DOPS/MO membranes containing high concentrations of DOPS were in the L(alpha) phase. First, the pH effect on the phase and structure of the multilamellar vesicles (MLVs) of the DOPS/MO membranes preformed at neutral pH was investigated by adding various low-pH buffers into the MLV suspension. For 20%-DOPS/80%-MO MLVs, at and below pH 2.9, a transition from the L(alpha) to cubic (Q(224)) phase occurred within 1 h. This phase transition was reversible; a subsequent increase in pH to a neutral one in the membrane suspension transformed the cubic phase into the original L(alpha) phase. Second, we found that a decrease in pH transformed large unilamellar vesicles of DOPS/MO membranes into the cubic phase under similar conditions. We have proposed the mechanism of the low-pH-induced phase transition and also made a quantitative analysis on the critical pH of the phase transition. This finding is the first demonstration that a change in pH can induce a reversible phase transition between the L(alpha) and cubic phases of lipid membranes within 1 h.
El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes.

Science.gov (United States)

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

1997-05-15

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

Science.gov (United States)

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

2007-10-15

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

Science.gov (United States)

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-28

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.
Hemi-fused structure mediates and controls fusion and fission in live cells.

Science.gov (United States)

Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

2016-06-23

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

Slaved diffusion in phospholipid bilayers

Science.gov (United States)

Zhang, Liangfang; Granick, Steve

2005-01-01

The translational diffusion of phospholipids in supported fluid bilayers splits into two populations when polyelectrolytes adsorb at incomplete surface coverage. Spatially resolved measurements using fluorescence correlation spectroscopy show that a slow mode, whose magnitude scales inversely with the degree of polymerization of the adsorbate, coexists with a fast mode characteristic of naked lipid diffusion. Inner and outer leaflets of the bilayer are affected nearly equally. Mobility may vary from spot to spot on the membrane surface, despite the lipid composition being the same. This work offers a mechanism to explain how nanosized domains with reduced mobility arise in lipid membranes. PMID:15967988
Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

Science.gov (United States)

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

2018-02-01

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.
Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

International Nuclear Information System (INIS)

Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming

2006-01-01

Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection
Lipid peroxidation and water penetration in lipid bilayers

DEFF Research Database (Denmark)

Conte, Elena; Megli, Francesco Maria; Khandelia, Himanshu

2012-01-01

to the hydroperoxide groups to interact with the nitroxide at the methyl-terminal, confirming that the H-bonds experimentally observed are due to increased water penetration in the bilayer. The EPR and MD data on model membranes demonstrate that cell membrane damage by oxidative stress cause alteration of water......(zz) parameters revealed that OHPLPC, but mostly HpPLPC, induced a measurable increase in polarity and H-bonding propensity in the central region of the bilayer. Molecular dynamics simulation performed on 16-DSA in the PLPC-HpPLPC bilayer revealed that water molecules are statistically favored with respect...
Viscoelastic deformation of lipid bilayer vesicles.

Science.gov (United States)

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

2015-10-07

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

Science.gov (United States)

Schoch, Rafael L.; Barel, Itay; Brown, Frank L. H.; Haran, Gilad

2018-03-01

Supported lipid bilayers (SLBs) have been studied extensively as simple but powerful models for cellular membranes. Yet, potential differences in the dynamics of the two leaflets of a SLB remain poorly understood. Here, using single particle tracking, we obtain a detailed picture of bilayer dynamics. We observe two clearly separate diffusing populations, fast and slow, that we associate with motion in the distal and proximal leaflets of the SLB, respectively, based on fluorescence quenching experiments. We estimate diffusion coefficients using standard techniques as well as a new method based on the blur of images due to motion. Fitting the observed diffusion coefficients to a two-leaflet membrane hydrodynamic model allows for the simultaneous determination of the intermonolayer friction coefficient and the substrate-membrane friction coefficient, without any prior assumptions on the strengths of the relevant interactions. Remarkably, our calculations suggest that the viscosity of the interfacial water confined between the membrane and the substrate is elevated by ˜104 as compared to bulk water. Using hidden Markov model analysis, we then obtain insight into the transbilayer movement of lipids. We find that lipid flip-flop dynamics are very fast, with half times in the range of seconds. Importantly, we find little evidence for membrane defect mediated lipid flip-flop for SLBs at temperatures well above the solid-to-liquid transition, though defects seem to be involved when the SLBs are cooled down. Our work thus shows that the combination of single particle tracking and advanced hydrodynamic modeling provides a powerful means to obtain insight into membrane dynamics.
Absorption and folding of melittin onto lipid bilayer membranes via unbiased atomic detail microsecond molecular dynamics simulation.

Science.gov (United States)

Chen, Charles H; Wiedman, Gregory; Khan, Ayesha; Ulmschneider, Martin B

2014-09-01

Unbiased molecular simulation is a powerful tool to study the atomic details driving functional structural changes or folding pathways of highly fluid systems, which present great challenges experimentally. Here we apply unbiased long-timescale molecular dynamics simulation to study the ab initio folding and partitioning of melittin, a template amphiphilic membrane active peptide. The simulations reveal that the peptide binds strongly to the lipid bilayer in an unstructured configuration. Interfacial folding results in a localized bilayer deformation. Akin to purely hydrophobic transmembrane segments the surface bound native helical conformer is highly resistant against thermal denaturation. Circular dichroism spectroscopy experiments confirm the strong binding and thermostability of the peptide. The study highlights the utility of molecular dynamics simulations for studying transient mechanisms in fluid lipid bilayer systems. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014. Published by Elsevier B.V.
Cholesterol enhances surface water diffusion of phospholipid bilayers

Energy Technology Data Exchange (ETDEWEB)

Cheng, Chi-Yuan; Kausik, Ravinath; Han, Songi, E-mail: songi@chem.ucsb.edu [Department of Chemistry and Biochemistry and Materials Research Laboratory, University of California, Santa Barbara, California 93106 (United States); Olijve, Luuk L. C. [Laboratory of Macromolecular and Organic Chemistry and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven (Netherlands)

2014-12-14

Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed {sup 1}H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5–10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroups or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in
Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers.

Science.gov (United States)

Mote, Kaustubh R; Gopinath, T; Traaseth, Nathaniel J; Kitchen, Jason; Gor'kov, Peter L; Brey, William W; Veglia, Gianluigi

2011-11-01

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring (1)H-(15)N dipolar couplings (DC) and (15)N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles' heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([(1)H,(15)N]-SE-PISEMA-PDSD). The incorporation of 2D (15)N/(15)N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the (15)N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers. © Springer Science+Business Media B.V. 2011
Interaction of saponin 1688 with phase separated lipid bilayers.

Science.gov (United States)

Chen, Maohui; Balhara, Vinod; Jaimes Castillo, Ana Maria; Balsevich, John; Johnston, Linda J

2017-07-01

Saponins are a diverse family of naturally occurring plant triterpene or steroid glycosides that have a wide range of biological activities. They have been shown to permeabilize membranes and in some cases membrane disruption has been hypothesized to involve saponin/cholesterol complexes. We have examined the interaction of steroidal saponin 1688-1 with lipid membranes that contain cholesterol and have a mixture of liquid-ordered (L o ) and liquid-disordered (L d ) phases as a model for lipid rafts in cellular membranes. A combination of atomic force microscopy (AFM) and fluorescence was used to probe the effect of saponin on the bilayer. The results demonstrate that saponin forms defects in the membrane and also leads to formation of small aggregates on the membrane surface. Although most of the membrane damage occurs in the liquid-disordered phase, fluorescence results demonstrate that saponin localizes in both ordered and disordered membrane phases, with a modest preference for the disordered regions. Similar effects are observed for both direct incorporation of saponin in the lipid mixture used to make vesicles/bilayers and for incubation of saponin with preformed bilayers. The results suggest that the initial sites of interaction are at the interface between the domains and surrounding disordered phase. The preference for saponin localization in the disordered phase may reflect the ease of penetration of saponin into a less ordered membrane, rather than the actual cholesterol concentration in the membrane. Dye leakage assays indicate that a high concentration of saponin is required for membrane permeabilization consistent with the supported lipid bilayer experiments. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.
Structure and physical properties of bio membranes and model membranes

International Nuclear Information System (INIS)

Tibor Hianik

2006-01-01

Bio membranes belong to the most important structures of the cell and the cell organelles. They play not only structural role of the barrier separating the external and internal part of the membrane but contain also various functional molecules, like receptors, ionic channels, carriers and enzymes. The cell membrane also preserves non-equilibrium state in a cell which is crucial for maintaining its excitability and other signaling functions. The growing interest to the bio membranes is also due to their unique physical properties. From physical point of view the bio membranes, that are composed of lipid bilayer into which are incorporated integral proteins and on their surface are anchored peripheral proteins and polysaccharides, represent liquid s crystal of smectic type. The bio membranes are characterized by anisotropy of structural and physical properties. The complex structure of bio membranes makes the study of their physical properties rather difficult. Therefore several model systems that mimic the structure of bio membranes were developed. Among them the lipid monolayers at an air-water interphase, bilayer lipid membranes, supported bilayer lipid membranes and liposomes are most known. This work is focused on the introduction into the physical word of the bio membranes and their models. After introduction to the membrane structure and the history of its establishment, the physical properties of the bio membranes and their models are stepwise presented. The most focus is on the properties of lipid monolayers, bilayer lipid membranes, supported bilayer lipid membranes and liposomes that were most detailed studied. This lecture has tutorial character that may be useful for undergraduate and graduate students in the area of biophysics, biochemistry, molecular biology and bioengineering, however it contains also original work of the author and his co-worker and PhD students, that may be useful also for specialists working in the field of bio membranes and model
Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

NARCIS (Netherlands)

Pecheur, EI; Martin, [No Value; Bienvenue, A; Ruysschaert, JM; Hoekstra, D

2000-01-01

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or
Thermodynamic free energy methods to investigate shape transitions in bilayer membranes.

Science.gov (United States)

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

2016-06-01

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

DEFF Research Database (Denmark)

Helfrich, W.; Thimmel, J.; Klösgen, Beate Maria

1999-01-01

The bilayers of some typical biological membrane lipids such as PC and DGDG disintegrate in a large excess of water to form an optically invisible dispersive bilayer phase. `Dark bodies' can be reversibly precipitated from it by raising the temperature. The dispersive phase probably consists...
Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

International Nuclear Information System (INIS)

Roche, Julien; Louis, John M.; Aniana, Annie; Ghirlando, Rodolfo; Bax, Ad

2015-01-01

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion
Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

Energy Technology Data Exchange (ETDEWEB)

Roche, Julien; Louis, John M.; Aniana, Annie [National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Chemical Physics (United States); Ghirlando, Rodolfo [National Institutes of Health, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Bax, Ad, E-mail: bax@nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Chemical Physics (United States)

2015-04-15

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.
Effects of Fatty Acid Inclusion in a DMPC Bilayer Membrane

DEFF Research Database (Denmark)

Peters, Günther H.J.; Hansen, Flemming Yssing; Møller, Martin S.

2009-01-01

Free fatty acids in biomembranes have been proposed to be a central component in several cellular control and regulatory mechanisms. To elucidate some fundamental elements underlying this, we have applied molecular dynamics simulations and experimental density measurements to study the molecular...... packing and structure of oleic acid (HOA) and stearic acid (HSA) in fluid bilayers of dimyristoylphosphatidylcholine (DMPC). The experimental data show a small but consistent positive excess volume for fatty acid concentrations below 10 mol %. At higher concentrations the fatty acids mix ideally...... with fluid DMPC. The simulations, which were benchmarked against the densitometric data, revealed interesting differences in the structure and location of the fatty acids depending on their protonation status. Thus, the protonated (uncharged) acid is located rather deeply in the membrane with an average...
Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

Science.gov (United States)

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.
Lipid bilayers suspended on microfabricated supports

Science.gov (United States)

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

2001-03-01

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

Science.gov (United States)

Ge, Chenhao; Orosz, Kristina S.; Armstrong, Neal R.; Saavedra, S. Scott

2011-01-01

Facilitated ion transport across an artificial lipid bilayer coupled to a solid substrate is a function common to several types of bioelectronic devices based on supported membranes, including biomimetic fuel cells and ion channel biosensors. Described here is fabrication of a pH-sensitive transducer composed of a porous sol-gel layer derivatized with poly(aniline) (PANI) nanowires grown from an underlying planar indium-tin oxide (ITO) electrode. The upper sol-gel surface is hydrophilic, smooth, and compatible with deposition of a planar supported lipid bilayer (PSLB) formed via vesicle fusion. Conducting tip AFM was used to show that the PANI wires are connected to the ITO, which convert this electrode into a potentiometric pH sensor. The response to changes in the pH of the buffer contacting the PANI nanowire/sol-gel/ITO electrode is blocked by the very low ion permeability of the overlying, fluid PSLB. The feasibility of using this assembly to monitor facilitated proton transport across the PSLB was demonstrated by doping the membrane with lipophilic ionophores that respond to a transmembrane pH gradient, which produced an apparent proton permeability several orders of magnitude greater than values measured for undoped lipid bilayers. PMID:21707069

Triglyceride Blisters in Lipid Bilayers: Implications for Lipid Droplet Biogenesis and the Mobile Lipid Signal in Cancer Cell Membranes

DEFF Research Database (Denmark)

Khandelia, Himanshu; Duelund, Lars; Pakkanen, Kirsi Inkeri

2010-01-01

triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid...... aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model...
Viscoelastic deformation of lipid bilayer vesicles†

Science.gov (United States)

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

2015-01-01

Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic. PMID:26268612
Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

Directory of Open Access Journals (Sweden)

de la Vega Michelle

2011-12-01

Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.
Formation of 3D cholesterol crystals from 2D nucleation sites in lipid bilayer membranes: implications for atherosclerosis.

Science.gov (United States)

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

2015-02-04

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

Science.gov (United States)

Hull, Marshall C; Cambrea, Lee R; Hovis, Jennifer S

2005-09-15

Infrared spectroscopy is a powerful technique for examining lipid bilayers; however, it says little about the fluidity of the bilayer-a key physical aspect. It is shown here that it is possible to both acquire spectroscopic data of supported lipid bilayer samples and make measurements of the membrane fluidity. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR) is used to obtain the spectroscopic information and fluorescence recovery after photobleaching (FRAP) is used to determine the fluidity of the samples. In the infrared spectra of lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the following major peaks were observed; nu(as)(CH3) 2954 cm(-1), nu(s)(CH3) 2870 cm(-1), nu(as)(CH2) 2924 cm(-1), nu(s)(CH2) 2852 cm(-1), nu(C=O) 1734 cm(-1), delta(CH2) 1463-1473 cm(-1), nu(as)(PO2-) 1226 cm(-1), nu(s)(PO2-) 1084 cm(-1), and nu(as)(N+(CH3)3) 973 cm(-1). The diffusion coefficient of the same lipid bilayer was measured to be 3.5 +/- 0.5 micom(2)/s with visual recovery also noted through use of epifluorescence microscopy. FRAP and visual data confirm the formation of a uniform, mobile supported lipid bilayer. The combination of ATR-FT-IR and FRAP provides complementary data giving a more complete picture of fully hydrated model membrane systems.
Alpha-tocopherol inhibits pore formation in oxidized bilayers

NARCIS (Netherlands)

Boonnoy, P.; Karttunen, M.; Wong-Ekkabut, J.

2017-01-01

In biological membranes, alpha-tocopherols (α-toc; vitamin E) protect polyunsaturated lipids from free radicals. Although the interactions of α-toc with non-oxidized lipid bilayers have been studied, their effects on oxidized bilayers remain unknown. In this study, atomistic molecular dynamics (MD)
Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

Directory of Open Access Journals (Sweden)

Jennifer S. Spence

2016-02-01

Full Text Available Ebola virus (EBOV makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1. GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveries, late events in EBOV entry following GP-NPC1 binding and culminating in GP-catalyzed fusion between viral and cellular lipid bilayers remain enigmatic. A mechanistic understanding of EBOV membrane fusion has been hampered by the failure of previous efforts to reconstitute fusion in vitro or at the cell surface. This report describes an assay to monitor initial steps directly in EBOV membrane fusion—triggering of GP and virus-cell lipid mixing—by single virions in live cells. Fusogenic triggering of GP occurs predominantly in Rab7-positive (Rab7+ endosomes, absolutely requires interaction between proteolytically primed GP and NPC1, and is blocked by key GP-specific neutralizing antibodies with therapeutic potential. Unexpectedly, cysteine protease inhibitors do not inhibit lipid mixing by virions bearing precleaved GP, even though they completely block cytoplasmic entry by these viruses, as shown previously. These results point to distinct cellular requirements for different steps in EBOV membrane fusion and suggest a model in which host cysteine proteases are dispensable for GP fusion triggering after NPC1 binding but are required for the formation of fusion pores that permit genome delivery.
Conformational Plasticity of the Influenza A M2 Transmembrane Helix in Lipid Bilayers Under Varying pH, Drug Binding and Membrane Thickness

Science.gov (United States)

Hu, Fanghao; Luo, Wenbin; Cady, Sarah D.; Hong, Mei

2010-01-01

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). 13C and 15N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, non-ideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and non-ideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (ϕ, ψ) torsion angles for three basis states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins. PMID:20883664
Simulations of skin barrier function: free energies of hydrophobic and hydrophilic transmembrane pores in ceramide bilayers.

Science.gov (United States)

Notman, Rebecca; Anwar, Jamshed; Briels, W J; Noro, Massimo G; den Otter, Wouter K

2008-11-15

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tail-vapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening.
Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

Directory of Open Access Journals (Sweden)

Mônica S Freitas

Full Text Available The Ebola fusion peptide (EBO₁₆ is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM, a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs, but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.
Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

Science.gov (United States)

Freitas, Mônica S; Follmer, Cristian; Costa, Lilian T; Vilani, Cecília; Bianconi, M Lucia; Achete, Carlos Alberto; Silva, Jerson L

2011-01-13

The Ebola fusion peptide (EBO₁₆) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.
Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

DEFF Research Database (Denmark)

Rønnest, A. K.; Peters, Günther H.J.; Hansen, Flemming Yssing

2016-01-01

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...... 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...
Lipid Bilayer Formation on Organic Electronic Materials

KAUST Repository

Zhang, Yi; Wustoni, Shofarul; Savva, Achilleas; Giovannitti, Alexander; McCulloch, Iain; Inal, Sahika

2018-01-01

The lipid bilayer is the elemental structure of cell membrane, forming a stable barrier between the interior and exterior of the cell while hosting membrane proteins that enable selective transport of biologically important compounds and cellular
Monitoring the Orientational Changes of Alamethicin during Incorporation into Bilayer Lipid Membranes.

Science.gov (United States)

Forbrig, Enrico; Staffa, Jana K; Salewski, Johannes; Mroginski, Maria Andrea; Hildebrandt, Peter; Kozuch, Jacek

2018-02-13

Antimicrobial peptides (AMPs) are the first line of defense after contact of an infectious invader, for example, bacterium or virus, with a host and an integral part of the innate immune system of humans. Their broad spectrum of biological functions ranges from cell membrane disruption over facilitation of chemotaxis to interaction with membrane-bound or intracellular receptors, thus providing novel strategies to overcome bacterial resistances. Especially, the clarification of the mechanisms and dynamics of AMP incorporation into bacterial membranes is of high interest, and different mechanistic models are still under discussion. In this work, we studied the incorporation of the peptaibol alamethicin (ALM) into tethered bilayer lipid membranes on electrodes in combination with surface-enhanced infrared absorption (SEIRA) spectroscopy. This approach allows monitoring the spontaneous and potential-induced ion channel formation of ALM in situ. The complex incorporation kinetics revealed a multistep mechanism that points to peptide-peptide interactions prior to penetrating the membrane and adopting the transmembrane configuration. On the basis of the anisotropy of the backbone amide I and II infrared absorptions determined by density functional theory calculations, we employed a mathematical model to evaluate ALM reorientations monitored by SEIRA spectroscopy. Accordingly, ALM was found to adopt inclination angles of ca. 69°-78° and 21° in its interfacially adsorbed and transmembrane incorporated states, respectively. These orientations can be stabilized efficiently by the dipolar interaction with lipid head groups or by the application of a potential gradient. The presented potential-controlled mechanistic study suggests an N-terminal integration of ALM into membranes as monomers or parallel oligomers to form ion channels composed of parallel-oriented helices, whereas antiparallel oligomers are barred from intrusion.
Sequential analysis of trans-SNARE formation in intracellular membrane fusion.

Directory of Open Access Journals (Sweden)

Kannan Alpadi

2012-01-01

Full Text Available SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a, Q(b, and Q(c from target (t-SNAREs and one (R from the vesicular (v-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a SNARE, leaving behind a Q(bcR subcomplex. This subcomplex serves as an acceptor for a Q(a SNARE from the opposite membrane, leading to Q(a-Q(bcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bcR cis-complex and the formation of the Q(a-Q(bcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex. This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.
Dynamics of a bilayer membrane coupled to a two-dimensional cytoskeleton: Scale transfers of membrane deformations

Science.gov (United States)

Okamoto, Ryuichi; Komura, Shigeyuki; Fournier, Jean-Baptiste

2017-07-01

We theoretically investigate the dynamics of a floating lipid bilayer membrane coupled with a two-dimensional cytoskeleton network, taking into account explicitly the intermonolayer friction, the discrete lattice structure of the cytoskeleton, and its prestress. The lattice structure breaks lateral continuous translational symmetry and couples Fourier modes with different wave vectors. It is shown that within a short time interval a long-wavelength deformation excites a collection of modes with wavelengths shorter than the lattice spacing. These modes relax slowly with a common renormalized rate originating from the long-wavelength mode. As a result, and because of the prestress, the slowest relaxation is governed by the intermonolayer friction. Conversely, and most interestingly, forces applied at the scale of the cytoskeleton for a sufficiently long time can cooperatively excite large-scale modes.
Physisorbed Polymer-Tethered Lipid Bilayer with Lipopolymer Gradient

Directory of Open Access Journals (Sweden)

Christoph A. Naumann

2012-11-01

Full Text Available Physisorbed polymer-tethered lipid bilayers consisting of phospholipids and lipopolymers represent an attractive planar model membrane platform, in which bilayer fluidity and membrane elastic properties can be regulated through lipopolymer molar concentration. Herein we report a method for the fabrication of such a planar model membrane system with a lateral gradient of lipopolymer density. In addition, a procedure is described, which leads to a sharp boundary between regions of low and high lipopolymer molar concentrations. Resulting gradients and sharp boundaries are visualized on the basis of membrane buckling structures at elevated lipopolymer concentrations using epifluorescence microscopy and atomic force microscopy. Furthermore, results from spot photobleaching experiments are presented, which provide insight into the lipid lateral fluidity in these model membrane architectures. The presented experimental data highlight a planar, solid-supported membrane characterized by fascinating length scale-dependent dynamics and elastic properties with remarkable parallels to those observed in cellular membranes.
Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

Science.gov (United States)

Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

2010-01-01

Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.
Sum frequency generation (SFG) vibrational spectroscopy of planar phosphatidylethanolamine hybrid bilayer membranes under water.

Science.gov (United States)

Kett, Peter J N; Casford, Michael T L; Davies, Paul B

2010-06-15

Sum frequency generation (SFG) spectroscopy has been used to study the structure of phosphatidylethanolamine hybrid bilayer membranes (HBMs) under water at ambient temperatures. The HBMs were formed using a modified Langmuir-Schaefer technique and consisted of a layer of dipalmitoyl phosphatidylethanolamine (DPPE) physisorbed onto an octadecanethiol (ODT) self-assembled monolayer (SAM) at a series of surface pressures from 1 to 40 mN m(-1). The DPPE and ODT were selectively deuterated so that the contributions to the SFG spectra from the two layers could be determined separately. SFG spectra in both the C-H and C-D stretching regions confirmed that a monolayer of DPPE had been adsorbed to the ODT SAM and that there were gauche defects within the alkyl chains of the phospholipid. On adsorption of a layer of DPPE, methylene modes from the ODT SAM were detected, indicating that the phospholipid had partially disordered the alkanethiol monolayer. SFG spectra recorded in air indicated that removal of water from the surface of the HBM resulted in disruption of the DPPE layer and the formation of phospholipid bilayers.
Development of an automation technique for the establishment of functional lipid bilayer arrays

International Nuclear Information System (INIS)

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

2009-01-01

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

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

Science.gov (United States)

Chen, Xiaoyun; Chen, Zhan

2006-09-01

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

Science.gov (United States)

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

2017-12-01

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

Energy Technology Data Exchange (ETDEWEB)

Poulos, J L [Librede Inc., Sherman Oaks, CA (United States); Portonovo, S A; Schmidt, J J [Department of Bioengineering, University of California, Los Angeles, Los Angeles (United States); Bang, H, E-mail: schmidt@seas.ucla.ed [School of Mechanical and Aerospace Engineering, Seoul National University (Korea, Republic of)

2010-11-17

Artificial lipid bilayer membranes have been used to reconstitute ion channels for scientific and technological applications. Membrane formation has traditionally involved slow, labor intensive processes best suited to small scale laboratory experimentation. We have recently demonstrated a high throughput method of membrane formation using automated liquid-handling robotics. We describe here the integration of membrane formation and measurement with two methods compatible with automation and high throughput liquid-handling robotics. Both of these methods create artificial lipid bilayers by joining lipid monolayers self-assembled at the interface of aqueous and organic phases using sessile aqueous droplets in contact with a measurement electrode; one using a pin tool, commonly employed in high throughput fluid handling assays, and the other using a positive displacement pipette. Membranes formed with both methods were high quality and supported measurement of ion channels at the single molecule level. Full automation of bilayer production and measurement with the positive displacement pipette was demonstrated by integrating it with a motion control platform.
Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

Science.gov (United States)

Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

2010-12-01

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.
Quantifying the Relationship Between Curvature and Electric Potential in Lipid Bilayers

DEFF Research Database (Denmark)

Bruhn, Dennis Skjøth; Lomholt, Michael Andersen; Khandelia, Himanshu

2016-01-01

Cellular membranes mediate vital cellular processes by being subject to curvature and transmembrane electrical potentials. Here we build upon the existing theory for flexoelectricity in liquid crystals to quantify the coupling between lipid bilayer curvature and membrane potentials. Using molecular...... dynamics simulations, we show that head group dipole moments, the lateral pressure profile across the bilayer and spontaneous curvature all systematically change with increasing membrane potentials. In particu- lar, there is a linear dependence between the bending moment (the product of bending rigidity...
Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer.

Science.gov (United States)

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

2005-12-01

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

International Nuclear Information System (INIS)

Jena, Bhanu P.

2009-01-01

The story of cell secretion and membrane fusion is as old as life itself. Without these fundamental cellular processes known to occur in yeast to humans, life would cease to exist. In the last 15 years, primarily using the atomic force microscope, a detailed understanding of the molecular process and of the molecular machinery and mechanism of secretion and membrane fusion in cells has come to light. This has led to a paradigm shift in our understanding of the underlying mechanism of cell secretion. The journey leading to the discovery of a new cellular structure the 'porosome',-the universal secretory machinery in cells, and the contributions of the AFM in our understanding of the general molecular machinery and mechanism of cell secretion and membrane fusion, is briefly discussed in this article.
Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

Science.gov (United States)

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

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino
The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

Science.gov (United States)

Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Schwartz, Cindi L; Fischer, Elizabeth R; Ryham, Rolf J; Cohen, Fredric S; Blank, Paul S; Zimmerberg, Joshua

2016-04-18

Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a 'lipidic junction'. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture-insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.
Regulation of membrane fusion and secretory events in the sea urchin embryo

International Nuclear Information System (INIS)

Roe, J.L.

1990-01-01

Membrane fusion and secretory events play a key role in fertilization and early development in the sea urchin embryo. To investigate the mechanism of membrane fusion, the effect of inhibitors of metalloendoprotease activity was studied on two model systems of cell fusion; fertilization and spiculogenesis by primary mesenchyme cells in the embryo. Both the zinc chelator, 1,10-phenanthroline, and peptide metalloprotease substrates were found to inhibit both fertilization and gamete fusion, while peptides that are not substrates of metalloproteases did not affect either process. Primary mesenchyme cells form the larval skeleton in the embryo by deposition of mineral and an organic matrix into a syncytial cavity formed by fusion of filopodia of these cells. Metalloprotease inhibitors were found to inhibit spiculogenesis both in vivo and in cultures of isolated primary mesenchyme cells, and the activity of a metalloprotease of the appropriate specificity was found in the primary mesenchyme cells. These two studies implicate the activity of a metalloprotease in a necessary step in membrane fusion. Following fertilization, exocytosis of the cortical granules results in the formation of the fertilization envelope and the hyaline layer, that surround the developing embryo. The hatching enzyme is secreted by the blastula stage sea urchin embryo, which proteolyzes the fertilization envelope surrounding the embryo, allowing the embryo to hatch. Using an assay that measures 125 I-fertilization envelope degradation, the hatching enzyme was identified as a 33 kDa metalloprotease, and was purified by ion-exchange and affinity chromatography from the hatching media of Strongylocentrotus purpuratus embryos. The hatching enzyme showed a substrate preference for only a minor subset of fertilization envelope proteins
Cholesterol effect on water permeability through DPPC and PSM lipid bilayers: a molecular dynamics study.

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Saito, Hiroaki; Shinoda, Wataru

2011-12-29

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

DEFF Research Database (Denmark)

Bai, M.; Miskowiec, A.; Hansen, F. Y.

2012-01-01

High-energy-resolution quasielastic neutron scattering has been used to elucidate the diffusion of water molecules in proximity to single bilayer lipid membranes supported on a silicon substrate. By varying sample temperature, level of hydration, and deuteration, we identify three different types...... of diffusive water motion: bulk-like, confined, and bound. The motion of bulk-like and confined water molecules is fast compared to those bound to the lipid head groups (7-10 H2O molecules per lipid), which move on the same nanosecond time scale as H atoms within the lipid molecules. Copyright (C) EPLA, 2012...
Plasmonic nanoantenna arrays for surface-enhanced Raman spectroscopy of lipid molecules embedded in a bilayer membrane.

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Kühler, Paul; Weber, Max; Lohmüller, Theobald

2014-06-25

We demonstrate a strategy for surface-enhanced Raman spectroscopy (SERS) of supported lipid membranes with arrays of plasmonic nanoantennas. Colloidal lithography refined with plasma etching is used to synthesize arrays of triangular shaped gold nanoparticles. Reducing the separation distance between the triangle tips leads to plasmonic coupling and to a strong enhancement of the electromagnetic field in the nanotriangle gap. As a result, the Raman scattering intensity of molecules that are located at this plasmonic "hot-spot" can be increased by several orders of magnitude. The nanoantenna array is then embedded with a supported phospholipid membrane which is fluid at room temperature and spans the antenna gap. This configuration offers the advantage that molecules that are mobile within the bilayer membrane can enter the "hot-spot" region via diffusion and can therefore be measured by SERS without static entrapment or adsorption of the molecules to the antenna itself.
Genomic analysis indicates the presence of an asymmetric bilayer outer membrane in Planctomycetes and Verrucomicrobia

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Daan R Speth

2012-08-01

Full Text Available Bacteria of the phylum Planctomycetes are of special interest for the study of compartmental cellular organization. Members of this phylum share a very unusual prokaryotic cell plan, featuring several membrane-bound compartments. Recently, it was shown that this cellular organization might extend to certain members of the phylum Verrucomicrobia. The Planctomycete cell plan has been defined as featuring a proteinaceous cell wall, a cytoplasmic membrane surrounding the paryphoplasm and an intracytoplasmic membrane defining the riboplasm. So far it was presumed that Planctomycetes did not have an asymmetric bilayer outer membrane as observed in Gram-negative bacteria. However, recent work on outer membrane biogenesis has provided several marker genes in the outer membrane protein (OMP assembly and the lipopolysaccharide (LPS insertion complexes. Additionally, advances in computational prediction of OMPs provided new tools to perform more accurate genomic screening for such proteins.Here we searched all 22 Planctomycetes and Verrucomicrobia genomes available in Genbank, plus the recently published genome of ‘Candidatus Scalindua profunda’, for markers of outer membrane biogenesis and OMPs. We were able to identify the key components of LPS insertion, OMP assembly and at least eight OMPs in all genomes tested. Additionally, we have analyzed the transcriptome and proteome data of the Planctomycetes ‘Candidatus Kuenenia stuttgartiensis’ and ‘Ca. S. profunda’ and could confirm high expression of several predicted OMPs, including the biomarkers of outer membrane biogenesis.
Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner

DEFF Research Database (Denmark)

Moesby, Lise; Corver, J; Erukulla, R K

1995-01-01

The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol ...
Alcohol's Effects on Lipid Bilayer Properties

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Ingólfsson, Helgi I.; Andersen, Olaf S.

2011-01-01

Alcohols are known modulators of lipid bilayer properties. Their biological effects have long been attributed to their bilayer-modifying effects, but alcohols can also alter protein function through direct protein interactions. This raises the question: Do alcohol's biological actions result predominantly from direct protein-alcohol interactions or from general changes in the membrane properties? The efficacy of alcohols of various chain lengths tends to exhibit a so-called cutoff effect (i.e., increasing potency with increased chain length, which that eventually levels off). The cutoff varies depending on the assay, and numerous mechanisms have been proposed such as: limited size of the alcohol-protein interaction site, limited alcohol solubility, and a chain-length-dependent lipid bilayer-alcohol interaction. To address these issues, we determined the bilayer-modifying potency of 27 aliphatic alcohols using a gramicidin-based fluorescence assay. All of the alcohols tested (with chain lengths of 1–16 carbons) alter the bilayer properties, as sensed by a bilayer-spanning channel. The bilayer-modifying potency of the short-chain alcohols scales linearly with their bilayer partitioning; the potency tapers off at higher chain lengths, and eventually changes sign for the longest-chain alcohols, demonstrating an alcohol cutoff effect in a system that has no alcohol-binding pocket. PMID:21843475
Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

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Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

2017-12-01

Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.
Coiled-coil formation of the membrane-fusion K/E peptides viewed by electron paramagnetic resonance.

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Pravin Kumar

Full Text Available The interaction of the complementary K (Ac-(KIAALKE3-GW-NH2 and E (Ac-(EIAALEK3-GY-NH2 peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009 is investigated by electron paramagnetic resonance (EPR. By frozen solution continuous-wave EPR and double electron-electron resonance (DEER, the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.
Proton permeation of lipid bilayers.

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Deamer, D W

1987-10-01

Proton permeation of the lipid bilayer barrier has two unique features. First, permeability coefficients measured at neutral pH ranges are six to seven orders of magnitude greater than expected from knowledge of other monovalent cations. Second, proton conductance across planar lipid bilayers varies at most by a factor of 10 when pH is varied from near 1 to near 11. Two mechanisms have been proposed to account for this anomalous behavior: proton conductance related to contaminants of lipid bilayers, and proton translocation along transient hydrogen-bonded chains (tHBC) of associated water molecules in the membrane. The weight of evidence suggests that trace contaminants may contribute to proton conductance across planar lipid membranes at certain pH ranges, but cannot account for the anomalous proton flux in liposome systems. Two new results will be reported here which were designed to test the tHBC model. These include measurements of relative proton/potassium permeability in the gramicidin channel, and plots of proton flux against the magnitude of pH gradients. (1) The relative permeabilities of protons and potassium through the gramicidin channel, which contains a single strand of hydrogen-bonded water molecules, were found to differ by at least four orders of magnitude when measured at neutral pH ranges. This result demonstrates that a hydrogen-bonded chain of water molecules can provide substantial discrimination between protons and other cations. It was also possible to calculate that if approximately 7% of bilayer water was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. (2) The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result that is consistent with one of three alternative tHBC models for proton conductance described by Nagle elsewhere in this volume.
Observation of undulation motion of lipid bilayers by neutron spin echo

International Nuclear Information System (INIS)

Yamada, Norifumi L.; Seto, Hideki; Hishida, Mafumi

2010-01-01

Aqueous solutions of synthesized phospholipids have been well investigated as model biomembranes. These lipids usually self-assemble into regular stacks of bilayers with a characteristic repeat distance on the order of nm, whereas real biomembrane exist as single bilayers. The key phenomenon in understanding the formation of single isolated bilayers in 'unbinding' of lipid bilayers, in which the inter-bilayer distance of lipid bilayers diverges by the steric interaction due to the membrane undulation. In this paper, we show some results of neutron spin-echo (NSE) experiments to investigate the effect of the steric interaction on unbinding and related phenomena. (author)

Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer.

Science.gov (United States)

Pluhackova, Kristyna; Wassenaar, Tsjerk A; Kirsch, Sonja; Böckmann, Rainer A

2015-03-26

A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.
Phytochemicals perturb membranes and promiscuously alter protein function.

Science.gov (United States)

Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

2014-08-15

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.
In vitro study of interaction of synaptic vesicles with lipid membranes

International Nuclear Information System (INIS)

Ghosh, S K; Castorph, S; Salditt, T; Konovalov, O; Jahn, R; Holt, M

2010-01-01

The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Although PIP 2 is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP 2 incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP 2 incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca 2+ ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.
A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

Science.gov (United States)

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.
Characterization of the transverse relaxation rates in lipid bilayers

International Nuclear Information System (INIS)

Watnick, P.I.; Dea, P.; Chan, S.I.

1990-01-01

The 2H NMR transverse relaxation rates of a deuterated phospholipid bilayer reflect slow motions in the bilayer membrane. A study of dimyristoyl lecithin specifically deuterated at several positions of the hydrocarbon chains indicates that these motions are cooperative and are confined to the hydrocarbon chains of the lipid bilayer. However, lipid head group interactions do play an important role in modulating the properties of the cooperative fluctuations of the hydrocarbon chains (director fluctuations), as evidenced by the effects of various lipid additives on the 2H NMR transverse relaxation rates of the dimyristoyl lecithin bilayer
Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

Science.gov (United States)

Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

1985-03-01

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.
Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

Science.gov (United States)

Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

2018-03-01

The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.
The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

Directory of Open Access Journals (Sweden)

Bin Lu

2015-06-01

Full Text Available Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2 on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26 abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9 loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.
Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

Science.gov (United States)

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

2014-11-01

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Manipulating lipid membrane architecture by liquid crystal-analog curvature elasticity (Presentation Recording)

Science.gov (United States)

Lee, Sin-Doo

2015-10-01

Soft matters such as liquid crystals and biological molecules exhibit a variety of interesting physical phenomena as well as new applications. Recently, in mimicking biological systems that have the ability to sense, regulate, grow, react, and regenerate in a highly responsive and self-adaptive manner, the significance of the liquid crystal order in living organisms, for example, a biological membrane possessing the lamellar order, is widely recognized from the viewpoints of physics and chemistry of interfaces and membrane biophysics. Lipid bilayers, resembling cell membranes, provide primary functions for the transport of biological components of ions and molecules in various cellular activities, including vesicle budding and membrane fusion, through lateral organization of the membrane components such as proteins. In this lecture, I will describe how the liquid crystal-analog curvature elasticity of a lipid bilayer plays a critical role in developing a new platform for understanding diverse biological functions at a cellular level. The key concept is to manipulate the local curvature at an interface between a solid substrate and a model membrane. Two representative examples will be demonstrated: one of them is the topographic control of lipid rafts in a combinatorial array where the ligand-receptor binding event occurs and the other concerns the reconstitution of a ring-type lipid raft in bud-mimicking architecture within the framework of the curvature elasticity.
Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating.

Science.gov (United States)

Nishikawa, Shuh-ichi; Hirata, Aiko; Endo, Toshiya

2008-11-01

During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.
Translocation of cell penetrating peptides and calcium-induced membrane fusion share same mechanism

Czech Academy of Sciences Publication Activity Database

Magarkar, Aniket; Allolio, Christoph; Jurkiewicz, Piotr; Baxová, Katarína; Šachl, Radek; Horinek, D.; Heinz, V.; Rachel, R.; Ziegler, C.; Jungwirth, Pavel

2017-01-01

Roč. 46, Suppl 1 (2017), S386 ISSN 0175-7571. [IUPAB congress /19./ and EBSA congress /11./. 16.07.2017-20.07.2017, Edinburgh] Institutional support: RVO:61388963 ; RVO:61388955 Keywords : membrane interactions * membrane fusion * cell penetration Subject RIV: BO - Biophysics
Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

International Nuclear Information System (INIS)

Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

2014-01-01

E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion
Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

Energy Technology Data Exchange (ETDEWEB)

Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

2014-05-15

E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.
Dynamic patterns in a supported lipid bilayer driven by standing surface acoustic waves.

Science.gov (United States)

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

2009-11-07

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

Directory of Open Access Journals (Sweden)

Christian Kleusch

2012-01-01

Full Text Available In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.
Potential and limitations of S-layers as support for planar lipid bilayers

International Nuclear Information System (INIS)

Kiene, E.

2011-01-01

then after chemical modification and no additional activation step was necessary. The lipid bilayer was fabricated by binding and fusion of discoidal lipid structures, so-called bicelles, while monitoring the process in AFM and QCM-D. As an exemplary transmembrane protein, the nicotinic acetylcholine receptor nAChR was isolated from T. californica and purified using magnetic beads coated with an nAChR specific antibody. An S-layer supported membrane could be fabricated by chemical adsorption and fusion of bicelles, but no bicelles could be bound using the Ni-HIS-interaction on the recombinant S-layer. Further crystallisation and Ni-HIS-tag binding studies, including TEM analysis, as well as near surface charge (zeta-potential) measurements of the inner and outer surface of SbpA lattices, gave interesting insights into the folding and crystallisation ability and the accessibility of the C-terminal HIS-tag of the recombinant protein. In conclusion, S-layer lattices were found to be a potentially powerful tool for nanobiotechnologic applications including the build-up of supported planar lipid membranes. However, great effort needs to be put into the thorough analysis of 3D structural models and subsequently into the optimisation of S-layer crystallisation processes and the accessibility of functional moieties. (author) [de
Phase separation in lipid bilayers triggered by low pH

International Nuclear Information System (INIS)

Suresh, Swetha; Edwardson, J. Michael

2010-01-01

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

International Nuclear Information System (INIS)

Pertsin, Alexander; Grunze, Michael

2014-01-01

Phospholipid bilayers supported on hydrophilic solids like silica and mica play a substantial role in fundamental studies and technological applications of phospholipid membranes. In both cases the molecular mechanism of adhesion between the bilayer and the support is of primary interest. Since the possibilities of experimental methods in this specific area are rather limited, the methods of computer simulation acquire great importance. In this paper we use the grand canonical Monte Carlo technique and an atomistic force field to simulate the behavior of a mica supported phospholipid bilayer in pure water as a function of the distance between the bilayer and the support. The simulation reveals a possible adhesion mechanism, where the adhesion is due to individual lipid molecules that protrude from the bilayer and form widely spaced links with the support. Simultaneously, the bilayer remains separated from the bilayer by a thin water interlayer which maintains the bilayer fluidity
Mutations that promote furin-independent growth of Semliki Forest virus affect p62-E1 interactions and membrane fusion

International Nuclear Information System (INIS)

Zhang Xinyong; Kielian, Margaret

2004-01-01

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1's fusion activity is regulated by its heterodimeric interaction with a companion membrane protein E2. Mature E2 protein is generated by furin processing of the precursor p62. Processing destabilizes the heterodimer, allowing dissociation at acidic pH, E1 conformational changes, and membrane fusion. We used a furin-deficient cell line, FD11, to select for SFV mutants that show increased growth in the absence of p62 processing. We isolated and characterized 7 such pci mutants (p62 cleavage independent), which retained the parental furin cleavage site but showed significant increases in their ability to carry out membrane fusion in the p62 form. Sequence analysis of the pci mutants identified mutations primarily on the E2 protein, and suggested sites important in the interaction of p62 with E1 and the regulation of fusion

Study on a multi-component palladium alloy membrane for the fusion fuel cycle

International Nuclear Information System (INIS)

Yoshida, Hiroshi; Okuno, Kenji; Nagasaki, Takanori; Noda, Kenji; Ishii, Yoshinobu; Takeshita, Hidefumi.

1985-11-01

A feasibility study on the material integrity with respect to the hydride formation and helium damage of the palladium alloy membrane was performed for an application of the palladium diffuser to a fusion fuel cleanup process. This study was conducted under the Japan/US Fusion Cooperation Program. Experimental works on the crystallography, hydrogen solubility and 3 He release characteristics were carried out with a multi-component palladium alloy(Pd-25Ag.Au.Ru). The excellent hydrogen permeability and mechanical properties of the membrane made of this alloy had been confirmed by authors' previous study. Based on the present study, this alloy membrane has high resistivity to the hydrogen embrittlement, and swelling and fracture due to the helium bubble formation under the practical operating conditions of the diffuser. (author)
Membrane support of accelerated fuel capsules for inertial fusion energy reactors

International Nuclear Information System (INIS)

Petzoldt, R.W.; Moir, R.W.

1993-01-01

The use of a thin membrane to suspend an (inertial fusion energy) fuel capsule in a holder for injection into a reactor chamber is investigated. Capsule displacement and membrane deformation angle are calculated for an axisymmetric geometry for a range of membrane strain and capsule size. This information is used to calculate maximum target accelerations. Membranes must be thin (perhaps of order one micron) to minimize their effect on capsule implosion symmetry. For example, a 5 μm thick cryogenic mylar membrane is calculated to allow 1,000 m/s 2 acceleration of a 3 mm radius, 100 mg capsule. Vibration analysis (for a single membrane support) shows that if membrane vibration is not deliberately minimized, allowed acceleration may be reduced by a factor of four. A two membrane alternative geometry would allow several times greater acceleration. Therefore, alternative membrane geometry's should be used to provide greater target acceleration potential and reduce capsule displacement within the holder (for a given membrane thickness)
Super-Sensitive and Robust Biosensors from Supported Polymer Bilayers

Energy Technology Data Exchange (ETDEWEB)

Paxton, Walter F. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)

2015-09-01

Biological organisms are potentially the most sensitive and selective biological detection systems known, yet we are currently severely limited in our ability to exploit biological interactions in sensory devices, due in part to the limited stability of biological systems and derived materials. This proposal addresses an important aspect of integrating biological sensory materials in a solid state device. If successful, such technology could enable entirely new classes of robust biosensors that could be miniaturized and deployed in the field. The critical aims of the proposed work were 1) the calibration of a more versatile approach to measuring pH, 2) the use of this method to monitor pH changes caused by the light-induced pumping of protons across vesicles with bacteriorhodopsin integrated into the membranes (either polymer or lipid); 3) the preparation of bilayer assemblies on platinum surfaces; 4) the enhanced detection of lightinduced pH changes driven by bR-loaded supported bilayers. I have developed a methodology that may enable that at interfaces and developed a methodology to characterize the functionality of bilayer membranes with reconstituted membrane proteins. The integrity of the supported bilayer films however must be optimized prior to the full realization of the work originally envisioned in the original proposal. Nevertheless, the work performed on this project and the encouraging results it has produced has demonstrated that these goals are challenging yet within reach.
Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

Science.gov (United States)

Lete, Marta G; Byrne, Richard D; Alonso, Alicia; Poccia, Dominic; Larijani, Banafshé

2017-01-15

Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P 3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P 3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes. © 2017. Published by The Company of Biologists Ltd.
A new look at lipid-membrane structure in relation to drug research

DEFF Research Database (Denmark)

Mouritsen, Ole G.; Jørgensen, Kent

1998-01-01

Lipid-bilayer membranes are key objects in drug research in relation to (i) interaction of drugs with membrane-bound receptors, (ii) drug targeting, penetration, and permeation of cell membranes, and (iii) use of liposomes in micro-encapsulation technologies for drug delivery. Rational design...... of new drugs and drug-delivery systems therefore requries insight into the physical properties of lipid-bilayer membranes. This mini-review provides a perspective on the current view of lipid-bilayer structure and dynamics based on information obtained from a variety of recent experimental...... and theoretical studies. Special attention is paid to trans-bilayer structure, lateral molecular organization of the lipid bilayer, lipid-mediated protein assembly, and lipid-bilayer permeability. It is argued that lipids play a major role in lipid membrane-organization and functionality....
Lipid Bilayer – mediated Regulation of Ion Channel Function by Amphiphilic Drugs

DEFF Research Database (Denmark)

Lundbæk, Jens August

2008-01-01

that are transforming it into a subject of quantitative science. It is described how the hydrophobic interactions between a membrane protein and the host lipid bilayer provide the basis for a mechanism, whereby protein function is regulated by the bilayer physical properties. The use of gramicidin channels as single-molecule......Drugs that at pico- to nanomolar concentration regulate ion channel function by high-affi nity binding to their cognate receptor often have a “ secondary pharmacology, ” in which the same molecule at low micromolar concentrations regulates a diversity of membrane proteins in an apparently...... nonspecifi c manner. It has long been suspected that this promiscuous regulation of membrane protein function could be due to changes in the physical properties of the host lipid bilayer, but the underlying mechanisms have been poorly understood. Given that pharmacological research often involves drug...
GABA_A receptor function is regulated by lipid bilayer elasticity

DEFF Research Database (Denmark)

Søgaard, Rikke; Werge, Thomas; Berthelsen, Camilla

2006-01-01

( s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABAA receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown...... reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABAA receptor function is regulated by lipid bilayer elasticity....... PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer....
In vitro study of interaction of synaptic vesicles with lipid membranes

Energy Technology Data Exchange (ETDEWEB)

Ghosh, S K; Castorph, S; Salditt, T [Institute for X-ray Physics, University of Goettingen, 37077 Goettingen (Germany); Konovalov, O [European Synchrotron Radiation Facility, 38043 Grenoble Cedex (France); Jahn, R; Holt, M, E-mail: sghosh1@gwdg.d, E-mail: mholt@gwdg.d, E-mail: tsaldit@gwdg.d [Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen (Germany)

2010-10-15

The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}). Although PIP{sub 2} is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP{sub 2} incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP{sub 2} incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca{sup 2+} ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.
Computer Simulations of Lipid Bilayers and Proteins

DEFF Research Database (Denmark)

Sonne, Jacob

2006-01-01

The importance of computer simulations in lipid bilayer research has become more prominent for the last couple of decades and as computers get even faster, simulations will play an increasingly important part of understanding the processes that take place in and across cell membranes. This thesis...... entitled Computer simulations of lipid bilayers and proteins describes two molecular dynamics (MD) simulation studies of pure lipid bilayers as well as a study of a transmembrane protein embedded in a lipid bilayer matrix. Below follows a brief overview of the thesis. Chapter 1. This chapter is a short...... in the succeeding chapters is presented. Details on system setups, simulation parameters and other technicalities can be found in the relevant chapters. Chapter 3, DPPC lipid parameters: The quality of MD simulations is intimately dependent on the empirical potential energy function and its parameters, i...
Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms.

Science.gov (United States)

Bhalla, Akhil; Chicka, Michael C; Chapman, Edwin R

2008-08-01

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.
Corrugation of Phase-Separated Lipid Bilayers Supported by Nanoporous Silica Xerogel Surfaces

Energy Technology Data Exchange (ETDEWEB)

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

2008-10-30

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

International Nuclear Information System (INIS)

Zidovetzki, R.; Banerjee, U.; Harrington, D.W.; Chan, S.I.

1988-01-01

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31 P and 2 H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers
Flunarizine Prevents Hepatitis C Virus Membrane Fusion in a Genotype-dependent Manner by Targeting the Potential Fusion Peptide within E1

Science.gov (United States)

Perin, Paula M.; Haid, Sibylle; Brown, Richard J. P.; Doerrbecker, Juliane; Schulze, Kai; Zeilinger, Carsten; von Schaewen, Markus; Heller, Brigitte; Vercauteren, Koen; Luxenburger, Eva; Baktash, Yasmine M.; Vondran, Florian W. R.; Speerstra, Sietkse; Awadh, Abdullah; Mukhtarov, Furkat; Schang, Luis M; Kirschning, Andreas; Müller, Rolf; Guzman, Carlos A.; Kaderali, Lars; Randall, Glenn; Meuleman, Philip; Ploss, Alexander; Pietschmann, Thomas

2015-01-01

To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies. PMID:26248546
Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Science.gov (United States)

Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

2009-02-01

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.
Influence of natural organic matter (NOM) coatings on nanoparticle adsorption onto supported lipid bilayers.

Science.gov (United States)

Bo, Zhang; Avsar, Saziye Yorulmaz; Corliss, Michael K; Chung, Minsub; Cho, Nam-Joon

2017-10-05

As the worldwide usage of nanoparticles in commercial products continues to increase, there is growing concern about the environmental risks that nanoparticles pose to biological systems, including potential damage to cellular membranes. A detailed understanding of how different types of nanoparticles behave in environmentally relevant conditions is imperative for predicting and mitigating potential membrane-associated toxicities. Herein, we investigated the adsorption of two popular nanoparticles (silver and buckminsterfullerene) onto biomimetic supported lipid bilayers of varying membrane charge (positive and negative). The quartz crystal microbalance-dissipation (QCM-D) measurement technique was employed to track the adsorption kinetics. Particular attention was focused on understanding how natural organic matter (NOM) coatings affect nanoparticle-bilayer interactions. Both types of nanoparticles preferentially adsorbed onto the positively charged bilayers, although NOM coatings on the nanoparticle and lipid bilayer surfaces could either inhibit or promote adsorption in certain electrolyte conditions. While past findings showed that NOM coatings inhibit membrane adhesion, our findings demonstrate that the effects of NOM coatings are more nuanced depending on the type of nanoparticle and electrolyte condition. Taken together, the results demonstrate that NOM coatings can modulate the lipid membrane interactions of various nanoparticles, suggesting a possible way to improve the environmental safety of nanoparticles. Copyright © 2017 Elsevier B.V. All rights reserved.
Neutron diffraction studies of amphipathic helices in phospholipid bilayers

Energy Technology Data Exchange (ETDEWEB)

Bradshaw, J.P.; Gilchrist, P.J. [Univ. of Edinburgh (United Kingdom); Duff, K.C. [Univ. of Edinburgh Medical School (United Kingdom); Saxena, A.M. [Brookhaven National Laboratory, Upton, NY (United States)

1994-12-31

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

International Nuclear Information System (INIS)

Bradshaw, J.P.; Gilchrist, P.J.; Duff, K.C.; Saxena, A.M.

1994-01-01

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

Science.gov (United States)

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

2008-10-28

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

DEFF Research Database (Denmark)

Venturoli, M.; Smit, B.; Sperotto, Maria Maddalena

2005-01-01

membranes. Here we present a mesoscopic model for lipid bilayers with embedded proteins, which we have studied with the help of the dissipative particle dynamics simulation technique. Because hydrophobic matching is believed to be one of the main physical mechanisms regulating lipid-protein interactions......-induced protein tilt, with the hydrophobic mismatch ( positive and negative) between the protein hydrophobic length and the pure lipid bilayer hydrophobic thickness. The protein-induced bilayer perturbation was quantified in terms of a coherence length, xi(P), of the lipid bilayer hydrophobic thickness pro. le...... for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting ( or overshooting) region where the bilayer hydrophobic thickness...
Acceleration of Lateral Equilibration in Mixed Lipid Bilayers Using Replica Exchange with Solute Tempering.

Science.gov (United States)

Huang, Kun; García, Angel E

2014-10-14

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

Molecular Transport Studies Through Unsupported Lipid Membranes

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Rock, William; Parekh, Sapun; Bonn, Mischa

2014-03-01

Dendrimers, spherical polymeric nanoparticles made from branched monomers around a central core, show great promise as drug delivery vehicles. Dendrimer size, core contents, and surface functionality can be synthetically tuned, providing unprecedented versatility. Polyamidoamine (PAMAM) dendrimers have been shown to enter cells; however, questions remain about their biophysical interactions with the cell membrane, specifically about the presence and size of transient pores. We monitor dendrimer-lipid bilayer interactions using unsupported black lipid membranes (BLMs) as model cell membranes. Custom bilayer slides contain two vertically stacked aqueous chambers separated by a 25 μm Teflon sheet with a 120 μm aperture where the bilayer is formed. We vary the composition of model membranes (cholesterol content and lipid phase) to create biomimetic systems and study the interaction of PAMAM G6 and G3 dendrimers with these bilayers. Dendrimers, dextran cargo, and bilayers are monitored and quantified using time-lapse fluorescence imaging. Electrical capacitance measurements are simultaneously recorded to determine if the membrane is porous, and the pore size is deduced by monitoring transport of fluorescent dextrans of increasing molecular weight. These experiments shed light on the importance of cholesterol content and lipid phase on the interaction of dendrimer nanoparticles with membranes.
Separating attoliter-sized compartments using fluid pore-spanning lipid bilayers.

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Lazzara, Thomas D; Carnarius, Christian; Kocun, Marta; Janshoff, Andreas; Steinem, Claudia

2011-09-27

Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55-60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5-15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging. © 2011 American Chemical Society
Influence of ester-modified lipids on bilayer structure.

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

2013-11-19

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

Science.gov (United States)

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.
Overcoming bottlenecks in the membrane protein structural biology pipeline.

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Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

2016-06-15

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
A model of lipid rearrangements during pore formation in the DPPC lipid bilayer.

Science.gov (United States)

Wrona, Artur; Kubica, Krystian

2017-07-10

The molecular bases of pore formation in the lipid bilayer remain unclear, as do the exact characteristics of their sizes and distributions. To understand this process, numerous studies have been performed on model lipid membranes including cell-sized giant unilamellar vesicles (GUV). The effect of an electric field on DPPC GUV depends on the lipid membrane state: in the liquid crystalline phase the created pores have a cylinder-like shape, whereas in the gel phase a crack has been observed. The aim of the study was to investigate the geometry of pores created in a lipid bilayer in gel and liquid crystalline phases in reference to literature experimental data. A mathematical model of the pore in a DPPC lipid bilayer developed based on the law of conservation of mass and the assumption of constant volume of lipid molecules, independent of their conformation, allows for analysis of pore shape and accompanying molecular rearrangements. The membrane area occupied by the pore of a cylinder-like shape is greater than the membrane area occupied by lipid molecules creating the pore structure (before pore appearance). Creation of such pores requires more space, which can be achieved by conformational changes of lipid chains toward a more compact state. This process is impossible for a membrane in the most compact, gel phase. We show that the geometry of the pores formed in the lipid bilayer in the gel phase must be different from the cylinder shape formed in the lipid bilayer in a liquid crystalline state, confirming experimental studies. Furthermore, we characterize the occurrence of the 'buffer' zone surrounding pores in the liquid crystalline phase as a mechanism of separation of neighbouring pores.
A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

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Matthew P Wood

Full Text Available Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
High yield, reproducible and quasi-automated bilayer formation in a microfluidic format

NARCIS (Netherlands)

Schulze Greiving-Stimberg, Verena Carolin; Bomer, Johan G.; van Uitert, I.; van den Berg, Albert; le Gac, Severine

2013-01-01

A microfluidic platform is reported for various experimentation schemes on cell membrane models and membrane proteins using a combination of electrical and optical measurements, including confocal microscopy. Bilayer lipid membranes (BLMs) are prepared in the device upon spontaneous and
The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

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Juan Fontana

2017-08-01

Full Text Available All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV, a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i mutagenesis to insert fluorescent proteins at specific positions, (ii coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.
Cholesterol Induced Changes in the Characteristics of the Time Series From Planar Lipid Bilayer Membrane during Electroporation

International Nuclear Information System (INIS)

Kotulska, M.; Koronkiewicz, S.; Kalinowski, S.

2002-01-01

The electroporation can be used as a non-toxic method for introducing exogenous macromolecules, especially DNA and drugs, into various types of cells. Research in to new therapeutic methods based on Long Duration Electroporation (LDE) is of special interest. A new current-clamp method makes possible the electroporation of very long duration with no damage to bio-membranes. In this paper we compare responses of lipid planar bilayer membranes at physiological concentration of KCl, with lipid membranes formed at higher ionic strength, and membranes containing cholesterol. A longer lifespan of the membranes with cholesterol and membranes with increased ionic strength could be observed. Sensitivity of the power spectrum response to the presence of cholesterol, ionic strength, current intensity, and membrane ageing was examined. The membrane memory was analyzed by means of autocorrelation function and rescaled range analysis. We showed that the memory of the system decreases for higher current intensities and this relation is pronounced better at higher ionic strength. At low current intensities all membranes showed slightly persistent type of noise behavior with crossover to Brownian type of noise for higher current value. The transition w as much faster for higher ionic strength, where the next transition to anti-persistent response was observed for relatively low currents. Very interesting results were obtained from power spectrum analysis. At low current intensity, all membranes exhibited 1/f noise, which disappeared for higher currents, maintaining f β type with rising value of β. Membranes formed at lower ionic strength and with cholesterol showed a pronounced tendency to lose flicker noise while ageing, also with rising β value. (author)
Confocal Raman Microscopy for in Situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles.

Science.gov (United States)

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

2018-06-05

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

Science.gov (United States)

Unsay, Joseph D; Cosentino, Katia; Subburaj, Yamunadevi; García-Sáez, Ana J

2013-12-23

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.
Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant

International Nuclear Information System (INIS)

Yao Yi; Ghosh, Kakoli; Epand, Raquel F.; Epand, Richard M.; Ghosh, Hara P.

2003-01-01

The fusogenic envelope glycoprotein G of the rhabdovirus vesicular stomatitis virus (VSV) induces membrane fusion at acidic pH. At acidic pH the G protein undergoes a major structural reorganization leading to the fusogenic conformation. However, unlike other viral fusion proteins, the low-pH-induced conformational change of VSV G is completely reversible. As well, the presence of an α-helical coiled-coil motif required for fusion by a number of viral and cellular fusion proteins was not predicted in VSV G protein by using a number of algorithms. Results of pH dependence of the thermal stability of G protein as determined by intrinsic Trp fluorescence and circular dichroism (CD) spectroscopy show that the G protein is equally stable at neutral or acidic pH. Destabilization of G structure at neutral pH with either heat or urea did not induce membrane fusion or conformational change(s) leading to membrane fusion. Taken together, these data suggest that the mechanism of VSV G-induced fusion is distinct from the fusion mechanism of fusion proteins that involve a coiled-coil motif
On calculation of the electrostatic potential of a phosphatidylinositol phosphate-containing phosphatidylcholine lipid membrane accounting for membrane dynamics.

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Jonathan C Fuller

Full Text Available Many signaling events require the binding of cytoplasmic proteins to cell membranes by recognition of specific charged lipids, such as phosphoinositol-phosphates. As a model for a protein-membrane binding site, we consider one charged phosphoinositol phosphate (PtdIns(3P embedded in a phosphatidylcholine bilayer. As the protein-membrane binding is driven by electrostatic interactions, continuum solvent models require an accurate representation of the electrostatic potential of the phosphoinositol phosphate-containing membrane. We computed and analyzed the electrostatic potentials of snapshots taken at regular intervals from molecular dynamics simulations of the bilayer. We observe considerable variation in the electrostatic potential of the bilayer both along a single simulation and between simulations performed with the GAFF or CHARMM c36 force fields. However, we find that the choice of GAFF or CHARMM c36 parameters has little effect on the electrostatic potential of a given configuration of the bilayer with a PtdIns(3P embedded in it. From our results, we propose a remedian averaging method for calculating the electrostatic potential of a membrane system that is suitable for simulations of protein-membrane binding with a continuum solvent model.
Controlling the shape of membrane protein polyhedra

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Li, Di; Kahraman, Osman; Haselwandter, Christoph A.

2017-03-01

Membrane proteins and lipids can self-assemble into membrane protein polyhedral nanoparticles (MPPNs). MPPNs have a closed spherical surface and a polyhedral protein arrangement, and may offer a new route for structure determination of membrane proteins and targeted drug delivery. We develop here a general analytic model of how MPPN self-assembly depends on bilayer-protein interactions and lipid bilayer mechanical properties. We find that the bilayer-protein hydrophobic thickness mismatch is a key molecular control parameter for MPPN shape that can be used to bias MPPN self-assembly towards highly symmetric and uniform MPPN shapes. Our results suggest strategies for optimizing MPPN shape for structural studies of membrane proteins and targeted drug delivery.
Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes.

OpenAIRE

Hoch, D H; Romero-Mira, M; Ehrlich, B E; Finkelstein, A; DasGupta, B R; Simpson, L L

1985-01-01

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These f...
Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

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Marina Gomzikova

2018-01-01

Full Text Available Extracellular vesicles (EV represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.
The influence of oscillating electromagnetic fields on membrane structure and function: Synthetic liposome and natural membrane bilayer systems with direct application to the controlled delivery of chemical agents

International Nuclear Information System (INIS)

Liburdy, R.P.; de Manincor, D.; Fingado, B.

1989-09-01

Investigations have been conducted to determine if an imposed electromagnetic field can influence membrane transport, and ion and drug permeability in both synthetic and natural cell membrane systems. Microwave fields enhance accumulation of sodium in the lymphocyte and induce protein shedding at Tc. Microwaves also trigger membrane permeability of liposome systems under specific field exposure conditions. Sensitivity varies in a defined way in bilayers displaying a membrane structural phase transition temperature, Tc; maximal release was observed at or near Tc. Significantly, liposome systems without a membrane phase transition were also found to experience permeability increases but, in contrast, this response was temperature independent. The above results indicate that field-enhanced drug release occurs in liposome vesicles that possess a Tc as well as non-Tc liposomes. Additional studies extend non-Tc liposome responses to the in vivo case in which microwaves trigger Gentamicin release from a liposome ''depot'' placed subcutaneously in the rat hind leg. In addition, evidence is provided that cell surface sequestered liposomes can be triggered by microwave fields to release drugs directly into target cells. 24 refs., 6 figs
Conformational study of the protegrin-1 (PG-1 dimer interaction with lipid bilayers and its effect

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Nussinov Ruth

2007-04-01

Full Text Available Abstract Background Protegrin-1 (PG-1 is known as a potent antibiotic peptide; it prevents infection via an attack on the membrane surface of invading microorganisms. In the membrane, the peptide forms a pore/channel through oligomerization of multiple subunits. Recent experimental and computational studies have increasingly unraveled the molecular-level mechanisms underlying the interactions of the PG-1 β-sheet motifs with the membrane. The PG-1 dimer is important for the formation of oligomers, ordered aggregates, and for membrane damaging effects. Yet, experimentally, different dimeric behavior has been observed depending on the environment: antiparallel in the micelle environment, and parallel in the POPC bilayer. The experimental structure of the PG-1 dimer is currently unavailable. Results Although the β-sheet structures of the PG-1 dimer are less stable in the bulk water environment, the dimer interface is retained by two intermolecular hydrogen bonds. The formation of the dimer in the water environment implies that the pathway of the dimer invasion into the membrane can originate from the bulk region. In the initial contact with the membrane, both the antiparallel and parallel β-sheet conformations of the PG-1 dimer are well preserved at the amphipathic interface of the lipid bilayer. These β-sheet structures illustrate the conformations of PG-1 dimer in the early stage of the membrane attack. Here we observed that the activity of PG-1 β-sheets on the bilayer surface is strongly correlated with the dimer conformation. Our long-term goal is to provide a detailed mechanism of the membrane-disrupting effects by PG-1 β-sheets which are able to attack the membrane and eventually assemble into the ordered aggregates. Conclusion In order to understand the dimeric effects leading to membrane damage, extensive molecular dynamics (MD simulations were performed for the β-sheets of the PG-1 dimer in explicit water, salt, and lipid bilayers
Interaction of Cecropin B with Zwitterionic and Negatively Charged Lipid Bilayers Immobilized at Gold Electrode Surface

International Nuclear Information System (INIS)

Juhaniewicz, Joanna; Szyk-Warszyńska, Lilianna; Warszyński, Piotr; Sęk, Sławomir

2016-01-01

Membranolytic properties of cationic antimicrobial peptide cecropin B were investigated using electrochemical techniques, atomic force microscopy and quartz crystal microbalance with dissipation monitoring. Two types of artificial lipid bilayers supported on gold electrode were used as model systems composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Chol) at 7:3 molar ratio and L-α-phosphatidylethanolamine (E. coli) (PE), L-α-phosphatidylglycerol sodium salt (E. coli) (PG) at 8:2 molar ratio. Thus the lipid content was intended to represent either mammalian or bacterial membrane respectively. Model bilayers were exposed to cecropin B at 1 μM concentration and the changes in bilayer structure, permeability and morphology were monitored as a function of time. We have found that cecropin B does not show any pronounced effect on POPC/Chol bilayer, while PE/PG system was strongly affected in the presence of the peptide. This observation suggests that cecropin B shows some selectivity with respect to lipid composition of the membrane. In case of PE/PG membrane, we have observed that peptide action involves electrostatically driven adsorption of the cecropin B at the top of the bilayer with simultaneous fluidization and swelling of the membrane. The latter may facilitate the rearrangement and insertion of the molecules into the core of the lipid bilayer, which leads to further rupture and degradation of the film through formation of mixed peptide-lipid aggregates.

Dependency of {gamma}-secretase complex activity on the structural integrity of the bilayer

Energy Technology Data Exchange (ETDEWEB)

Zhou, Hua, E-mail: hzhou2@lbl.gov [Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 (United States); Zhou, Shuxia; Walian, Peter J.; Jap, Bing K. [Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 (United States)

2010-11-12

Research highlights: {yields} Partial solubilization of membranes with CHAPSO can increase {gamma}-secretase activity. {yields} Completely solubilized {gamma}-secretase is inactive. {yields} Purified {gamma}-secretase regains activity after reconstitution into lipid bilayers. {yields} A broad range of detergents can be used to successfully reconstitute {gamma}-secretase. -- Abstract: {gamma}-secretase is a membrane protein complex associated with the production of A{beta} peptides that are pathogenic in Alzheimer's disease. We have characterized the activity of {gamma}-secretase complexes under a variety of detergent solubilization and reconstitution conditions, and the structural state of proteoliposomes by electron microscopy. We found that {gamma}-secretase activity is highly dependent on the physical state or integrity of the membrane bilayer - partial solubilization may increase activity while complete solubilization will abolish it. The activity of well-solubilized {gamma}-secretase can be restored to near native levels when properly reconstituted into a lipid bilayer environment.
Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy.

Science.gov (United States)

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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

Science.gov (United States)

Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

2016-03-01

Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.
Henipavirus Mediated Membrane Fusion, Virus Entry and Targeted Therapeutics

Directory of Open Access Journals (Sweden)

Dimitar B. Nikolov

2012-02-01

Full Text Available The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G and class I fusion (F envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins.
Ion dynamics in cationic lipid bilayer systems in saline solutions

DEFF Research Database (Denmark)

Miettinen, Markus S; Gurtovenko, Andrey A; Vattulainen, Ilpo

2009-01-01

Positively charged lipid bilayer systems are a promising class of nonviral vectors for safe and efficient gene and drug delivery. Detailed understanding of these systems is therefore not only of fundamental but also of practical biomedical interest. Here, we study bilayers comprising a binary...... are concluded to be interesting for the physics of the whole membrane, especially considering its interaction dynamics with charged macromolecular surfaces....
Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

Science.gov (United States)

Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

2009-11-01

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.
Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide

Science.gov (United States)

Lousa, Diana; Pinto, Antónia R. T.; Victor, Bruno L.; Laio, Alessandro; Veiga, Ana S.; Castanho, Miguel A. R. B.; Soares, Cláudio M.

2016-01-01

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies. PMID:27302370
Large scale biomimetic membrane arrays

DEFF Research Database (Denmark)

Hansen, Jesper Søndergaard; Perry, Mark; Vogel, Jörg

2009-01-01

To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical-electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO2 laser micro......-structured 8 x 8 aperture partition arrays with average aperture diameters of 301 +/- 5 mu m. We addressed the electro-physical properties of the lipid bilayers established across the micro-structured scaffold arrays by controllable reconstitution of biotechnological and physiological relevant membrane...... peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24 x 24 and hexagonal 24 x 27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays...
Aggregation of Aß(25-35 on DOPC and DOPC/DHA bilayers: an atomic force microscopy study.

Directory of Open Access Journals (Sweden)

Matilde Sublimi Saponetti

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

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Pablo Guardado-Calvo

2016-10-01

Full Text Available Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS or of hantavirus pulmonary syndrome (HPS, depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.
Human serum albumin supported lipid patterns for the targeted recognition of microspheres coated by membrane based on ss-DNA hybridization

International Nuclear Information System (INIS)

Zhang Xiaoming; He Qiang; Cui Yue; Duan Li; Li Junbai

2006-01-01

Human serum albumin (HSA) patterns have been successfully fabricated for the deposition of lipid bilayer, 1,2-dimyristoyl-sglycerophosphate (DMPA), by making use of the micro-contact printing (μCP) technique and liposome fusion. Confocal laser scanning microscopy (CLSM) results indicate that lipid bilayer has been assembled in HSA patterns with a good stability. Such well-defined lipid patterns formed on HSA surface create possibility to incorporate specific components like channels or receptors for specific recognition. In view of this, microspheres coated with lipid membranes were immobilized in HSA-supported lipid patterns via the hybridization of complementary ss-DNAs. This procedure enables to transfer solid materials to a soft surface through a specific recognition
Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

Energy Technology Data Exchange (ETDEWEB)

Yu, Qianlong [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Blissard, Gary W. [Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, United State (United States); Liu, Tong-Xian [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Li, Zhaofei, E-mail: zhaofeili73@outlook.com [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China)

2016-01-15

The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.
Challenges in the Development of Functional Assays of Membrane Proteins

Directory of Open Access Journals (Sweden)

Sophie Demarche

2012-11-01

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

International Nuclear Information System (INIS)

Tu, Z C; Ou-Yang, Z C

2004-01-01

The purpose of this paper is to study the shapes and stabilities of bio-membranes within the framework of exterior differential forms. After a brief review of the current status of theoretical and experimental studies on the shapes of bio-membranes, a geometric scheme is proposed to discuss the shape equation of closed lipid bilayers, the shape equation and boundary conditions of open lipid bilayers and two-component membranes, the shape equation and in-plane strain equations of cell membranes with cross-linking structures, and the stabilities of closed lipid bilayers and cell membranes. The key point of this scheme is to deal with the variational problems on surfaces embedded in three-dimensional Euclidean space by using exterior differential forms
Asymmetric bi-layer PFSA membranes as model systems for the study of water management in the PEMFC.

Science.gov (United States)

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

2014-10-14

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

Energy Technology Data Exchange (ETDEWEB)

Pera, H.; Kleijn, J. M.; Leermakers, F. A. M., E-mail: Frans.leermakers@wur.nl [Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6307 HB Wageningen (Netherlands)

2014-02-14

To understand how lipid architecture determines the lipid bilayer structure and its mechanics, we implement a molecularly detailed model that uses the self-consistent field theory. This numerical model accurately predicts parameters such as Helfrichs mean and Gaussian bending modulus k{sub c} and k{sup ¯} and the preferred monolayer curvature J{sub 0}{sup m}, and also delivers structural membrane properties like the core thickness, and head group position and orientation. We studied how these mechanical parameters vary with system variations, such as lipid tail length, membrane composition, and those parameters that control the lipid tail and head group solvent quality. For the membrane composition, negatively charged phosphatidylglycerol (PG) or zwitterionic, phosphatidylcholine (PC), and -ethanolamine (PE) lipids were used. In line with experimental findings, we find that the values of k{sub c} and the area compression modulus k{sub A} are always positive. They respond similarly to parameters that affect the core thickness, but differently to parameters that affect the head group properties. We found that the trends for k{sup ¯} and J{sub 0}{sup m} can be rationalised by the concept of Israelachivili's surfactant packing parameter, and that both k{sup ¯} and J{sub 0}{sup m} change sign with relevant parameter changes. Although typically k{sup ¯}<0, membranes can form stable cubic phases when the Gaussian bending modulus becomes positive, which occurs with membranes composed of PC lipids with long tails. Similarly, negative monolayer curvatures appear when a small head group such as PE is combined with long lipid tails, which hints towards the stability of inverse hexagonal phases at the cost of the bilayer topology. To prevent the destabilisation of bilayers, PG lipids can be mixed into these PC or PE lipid membranes. Progressive loading of bilayers with PG lipids lead to highly charged membranes, resulting in J{sub 0}{sup m}≫0, especially at low ionic
Automatically Identifying Fusion Events between GLUT4 Storage Vesicles and the Plasma Membrane in TIRF Microscopy Image Sequences

Directory of Open Access Journals (Sweden)

Jian Wu

2015-01-01

Full Text Available Quantitative analysis of the dynamic behavior about membrane-bound secretory vesicles has proven to be important in biological research. This paper proposes a novel approach to automatically identify the elusive fusion events between VAMP2-pHluorin labeled GLUT4 storage vesicles (GSVs and the plasma membrane. The differentiation is implemented to detect the initiation of fusion events by modified forward subtraction of consecutive frames in the TIRFM image sequence. Spatially connected pixels in difference images brighter than a specified adaptive threshold are grouped into a distinct fusion spot. The vesicles are located at the intensity-weighted centroid of their fusion spots. To reveal the true in vivo nature of a fusion event, 2D Gaussian fitting for the fusion spot is used to derive the intensity-weighted centroid and the spot size during the fusion process. The fusion event and its termination can be determined according to the change of spot size. The method is evaluated on real experiment data with ground truth annotated by expert cell biologists. The evaluation results show that it can achieve relatively high accuracy comparing favorably to the manual analysis, yet at a small fraction of time.
The Integrin Receptor in Biologically Relevant Bilayers

DEFF Research Database (Denmark)

Kalli, Antreas C.; Róg, Tomasz; Vattulainen, Ilpo

2017-01-01

/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study...... demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin....../talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction...
ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

Science.gov (United States)

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.
Subterahertz Longitudinal Phonon Modes Propagating in a Lipid Bilayer Immersed in an Aqueous Medium

Science.gov (United States)

Zakhvataev, V. E.

2018-04-01

The properties of subterahertz longitudinal acoustic phonon modes in the hydrophobic region of a lipid bilayer immersed in a compressible viscous aqueous medium are investigated theoretically. An approximate expression is obtained for the Mandelstam-Brillouin components of the dynamic structure factor of a bilayer. The analysis is based on a generalized hydrodynamic model of the "two-dimensional lipid bilayer + three-dimensional fluid medium" system, as well as on known sharp estimates for the frequencies and lifetimes of long-wavelength longitudinal acoustic phonons in a free hydrated lipid bilayer and in water, obtained from inelastic X-ray scattering experiments and molecular dynamics simulations. It is shown that, for characteristic values of the parameters of the membrane system, subterahertz longitudinal phonon-like excitations in the hydrophobic part of the bilayer are underdamped. In this case, the contribution of the viscous flow of the aqueous medium to the damping of a longitudinal membrane mode is small compared with the contribution of the lipid bilayer. Quantitative estimates of the damping ratio agree well with the experimental results for the vibration mode of the enzyme lysozyme in aqueous solution [1]. It is also shown that a coupling between longitudinal phonon modes of the bilayer and relaxation processes in its fluid environment gives rise to an additional peak in the scattering spectrum, which corresponds to a non-propagating mode.

Effects of Lateral and Terminal Chains of X-Shaped Bolapolyphiles with Oligo(phenylene ethynylene Cores on Self-Assembly Behavior. Part 2: Domain Formation by Self-Assembly in Lipid Bilayer Membranes

Directory of Open Access Journals (Sweden)

Stefan Werner

2017-09-01

Full Text Available Supramolecular self-assembly of membrane constituents within a phospholipid bilayer creates complex functional platforms in biological cells that operate in intracellular signaling, trafficking and membrane remodeling. Synthetic polyphilic compounds of macromolecular or small size can be incorporated into artificial phospholipid bilayers. Featuring three or four moieties of different philicities, they reach beyond ordinary amphiphilicity and open up avenues to new functions and interaction concepts. Here, we have incorporated a series of X-shaped bolapolyphiles into DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers of giant unilamellar vesicles. The bolapolyphiles consist of a rod-like oligo(phenylene ethynylene (OPE core, hydrophilic glycerol-based headgroups with or without oligo(ethylene oxide expansions at both ends and two lateral alkyl chains attached near the center of the OPE core. In the absence of DPPC and water, the compounds showed thermotropic liquid-crystalline behavior with a transition between polyphilic and amphiphilic assembly (see part 1 in this issue. In DPPC membranes, various trends in the domain morphologies were observed upon structure variations, which entailed branched alkyl chains of various sizes, alkyl chain semiperfluorination and size expansion of the headgroups. Observed effects on domain morphology are interpreted in the context of the bulk behavior (part 1 and of a model that was previously developed based on spectroscopic and physicochemical data.
Cell fusion and nuclear fusion in plants.

Science.gov (United States)

Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

2016-12-01

Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.
Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores.

Science.gov (United States)

Gaede, Holly C; Luckett, Keith M; Polozov, Ivan V; Gawrisch, Klaus

2004-08-31

Lipid bilayers were deposited inside the 0.2 microm pores of anodic aluminum oxide (AAO) filters by extrusion of multilamellar liposomes and their properties studied by 2H, 31P, and 1H solid-state NMR. Only the first bilayer adhered strongly to the inner surface of the pores. Additional layers were washed out easily by a flow of water as demonstrated by 1H magic angle spinning NMR experiments with addition of Pr3+ ions to shift accessible lipid headgroup resonances. A 13 mm diameter Anopore filter of 60 microm thickness oriented approximately 2.5 x 10(-7) mol of lipid as a single bilayer, corresponding to a total membrane area of about 500 cm2. The 2H NMR spectra of chain deuterated POPC are consistent with adsorption of wavy, tubular bilayers to the inner pore surface. By NMR diffusion experiments, we determined the average length of those lipid tubules to be approximately 0.4 microm. There is evidence for a thick water layer between lipid tubules and the pore surface. The ends of tubules are well sealed against the pore such that Pr3+ ions cannot penetrate into the water underneath the bilayers. We successfully trapped poly(ethylene glycol) (PEG) with a molecular weight of 8000 in this water layer. From the quantity of trapped PEG, we calculated an average water layer thickness of 3 nm. Lipid order parameters and motional properties are unperturbed by the solid support, in agreement with existence of a water layer. Such unperturbed, solid supported membranes are ideal for incorporation of membrane-spanning proteins with large intra- and extracellular domains. The experiments suggest the promise of such porous filters as membrane support in biosensors.
A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles

DEFF Research Database (Denmark)

Etzerodt, Thomas P.; Trier, Sofie; Henriksen, Jonas R.

2012-01-01

sporadic and there is a strong need to characterize and increase our understanding of the membrane fusion properties of these peptides. Many fusion studies have focused on the ability of free peptides in solution that mediate fusion between liposomes. For drug delivery purposes it is a necessity to attach......,2-diamino propanoic acid (Dap) moiety, yielding the lipopeptide dimyristoyl-Dap-GALA (DMDGALA). We have investigated DMDGALA as a component in large unilamellar vesicles (LUVs) and demonstrate pH-triggered fusion of peptide containing LUVs with stable target giant unilamellar vesicles (GUVs), which were...
Geometric theory on the elasticity of bio-membranes

OpenAIRE

Tu, Z. C.; Ou-Yang, Z. C.

2004-01-01

The purpose of this paper is to study the shapes and stabilities of bio-membranes within the framework of exterior differential forms. After a brief review of the current status in theoretical and experimental studies on the shapes of bio-membranes, a geometric scheme is proposed to discuss the shape equation of closed lipid bilayers, the shape equation and boundary conditions of open lipid bilayers and two-component membranes, the shape equation and in-plane strain equations of cell membrane...
New optical method for measuring the bending elasticity of lipid bilayers

International Nuclear Information System (INIS)

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

2016-01-01

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

International Nuclear Information System (INIS)

Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M.

2015-01-01

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales
Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

Energy Technology Data Exchange (ETDEWEB)

Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

2015-04-15

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.
Nanodisc-solubilized membrane protein library reflects the membrane proteome

OpenAIRE

Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

2013-01-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...
Formation of supported lipid bilayers containing phase-segregated domains and their interaction with gold nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Melby, Eric S.; Mensch, Arielle C.; Lohse, Samuel E.; Hu, Dehong; Orr, Galya; Murphy, Catherine J.; Hamers, Robert J.; Pedersen, Joel A.

2016-01-01

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

Energy Technology Data Exchange (ETDEWEB)

Sathiyamoorthy, Karthik; Jiang, Jiansen; Möhl, Britta S.; Chen, Jia; Zhou, Z. Hong; Longnecker, Richard; Jardetzky, Theodore S. (UCLA); (Stanford-MED); (NWU)

2017-09-22

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studied the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.
Flavonoid-membrane Interactions: A Protective Role of Flavonoids at the Membrane Surface?

Directory of Open Access Journals (Sweden)

Patricia I. Oteiza

2005-01-01

Full Text Available Flavonoids can exert beneficial health effects through multiple mechanisms. In this paper, we address the important, although not fully understood, capacity of flavonoids to interact with cell membranes. The interactions of polyphenols with bilayers include: (a the partition of the more non-polar compounds in the hydrophobic interior of the membrane, and (b the formation of hydrogen bonds between the polar head groups of lipids and the more hydrophilic flavonoids at the membrane interface. The consequences of these interactions are discussed. The induction of changes in membrane physical properties can affect the rates of membrane lipid and protein oxidation. The partition of certain flavonoids in the hydrophobic core can result in a chain breaking antioxidant activity. We suggest that interactions of polyphenols at the surface of bilayers through hydrogen bonding, can act to reduce the access of deleterious molecules (i.e. oxidants, thus protecting the structure and function of membranes.
Exploring the membrane fusion mechanism through force-induced disassembly of HIV-1 six-helix bundle

Energy Technology Data Exchange (ETDEWEB)

Gao, Kai [Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Yong [Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Lou, Jizhong, E-mail: jlou@ibp.ac.cn [Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

2016-05-13

Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminal and C-terminal helix. -- Highlights: •Unfolding of HIV-1 gp41 six-helix bundle is studied by molecular dynamics simulations. •Specific interactions responsible for the stability of HIV-1 envelope post-fusion conformation were identified. •The gp41 six-helix bundle transition inducing membrane fusion might be a cooperative process of the three subunits.
Poly(amidoamine) dendrimers on lipid bilayers II: Effects of bilayer phase and dendrimer termination.

Science.gov (United States)

Kelly, Christopher V; Leroueil, Pascale R; Orr, Bradford G; Banaszak Holl, Mark M; Andricioaei, Ioan

2008-08-07

The molecular structures and enthalpy release of poly(amidoamine) (PAMAM) dendrimers binding to 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayers were explored through atomistic molecular dynamics. Three PAMAM dendrimer terminations were examined: protonated primary amine, neutral acetamide, and deprotonated carboxylic acid. Fluid and gel lipid phases were examined to extract the effects of lipid tail mobility on the binding of generation-3 dendrimers, which are directly relevant to the nanoparticle interactions involving lipid rafts, endocytosis, lipid removal, and/or membrane pores. Upon binding to gel phase lipids, dendrimers remained spherical, had a constant radius of gyration, and approximately one-quarter of the terminal groups were in close proximity to the lipids. In contrast, upon binding to fluid phase bilayers, dendrimers flattened out with a large increase in their asphericity and radii of gyration. Although over twice as many dendrimer-lipid contacts were formed on fluid versus gel phase lipids, the dendrimer-lipid interaction energy was only 20% stronger. The greatest enthalpy release upon binding was between the charged dendrimers and the lipid bilayer. However, the stronger binding to fluid versus gel phase lipids was driven by the hydrophobic interactions between the inner dendrimer and lipid tails.
Phase behavior of supported lipid bilayers: A systematic study by coarse-grained molecular dynamics simulations

DEFF Research Database (Denmark)

Poursoroush, Asma; Sperotto, Maria Maddalena; Laradji, Mohamed

2017-01-01

Solid-supported lipid bilayers are utilized by experimental scientists as models for biological membranes because of their stability. However, compared to free standing bilayers, their close proximity to the substrate may affect their phase behavior. As this is still poorly understood, and few co...
Rationalization of reduced penetration of drugs through ceramide gel phase membrane.

Science.gov (United States)

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

2014-11-25

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

Science.gov (United States)

Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

2014-06-01

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.
Water distribution function across the curved lipid bilayer: SANS study

International Nuclear Information System (INIS)

Kiselev, M.A.; Zemlyanaya, E.V.; Ryabova, N.Y.; Hauss, T.; Dante, S.; Lombardo, D.

2008-01-01

The neutron scattering length density across the membrane is simulated on the basis of fluctuated model of lipid bilayer. The use of a separated form factors method has been applied for the identification of the structural features of the polydispersed unilamellar DMPC vesicle system. The hydration of vesicle is described by sigmoid distribution function of the water molecules. The application of the model to the obtained SANS spectra allow the determination of the main parameters of the system, such as the average vesicle radius (and its polydispersity), the membrane thickness, the thickness of hydrocarbon chain region, the number of water molecules located per lipid molecule, and the phospholipid surface area. Moreover the approach allow the calculation of some relevant parameters connected with the water distribution function across the bilayer system. The main features of the obtained results furnish an explanation of why lipid membrane is easily penetrated by the water molecules of the solution
Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and {sup 13}C-{sup 2}H rotational-echo double-resonance solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Xie Li; Ghosh, Ujjayini; Schmick, Scott D.; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

2013-01-15

Residue-specific location of peptides in the hydrophobic core of membranes was examined using {sup 13}C-{sup 2}H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of {beta} sheet HIV and helical and {beta} sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.
The radiation effects on lipid bilayers

International Nuclear Information System (INIS)

Ikigai, Hajime; Matsuura, Tomio; Narita, Noboru; Ozawa, Atsushi.

1980-01-01

The Radiation effects on lipid bilayers are studied by the electron spin resonance. Egg lecithin liposomes and human erythrocytes are labeled with spin probes (5 SAL, 12 SAL). Effects of membrane fluidity by X-Ray (or ultraviolet) irradiation are measured by change of the order parameter S. The results obtained are as follows: 1) A similar tendency is observed on the order parameter S between X-Ray irradiated egg lecithin liposomes and human erythrocytes. 2) The rapid changes of the membrane fluidity are observed below 1 krad. The fluctuation of membrane fluidity decreases above 1 krad, consequently the membrane has a tendency changing to a rigid state at low dose area. 3) It is suggested that the more effective radicals are hydroxyl radicals and superoxide radicals. 4) The effects of ultraviolet irradiation with hydrogen peroxide show that hydroxyl radicals lead to changes of membrane fluidity. (author)

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

International Nuclear Information System (INIS)

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis

2006-01-01

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1 V12 or Cdc42 V12 could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA L63 decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia
Characteristics of TiO_2/ZnO bilayer film towards pH sensitivity prepared by different spin coating deposition process

International Nuclear Information System (INIS)

Rahman, Rohanieza Abdul; Zulkefle, Muhammad Al Hadi; Abdullah, Wan Fazlida Hanim; Rusop, M.; Herman, Sukreen Hana

2016-01-01

In this study, titanium dioxide (TiO_2) and zinc oxide (ZnO) bilayer film for pH sensing application will be presented. TiO_2/ZnO bilayer film with different speed of spin-coating process was deposited on Indium Tin Oxide (ITO), prepared by sol-gel method. This fabricated bilayer film was used as sensing membrane for Extended Gate Field-Effect Transistor (EGFET) for pH sensing application. Experimental results indicated that the sensor is able to detect the sensitivity towards pH buffer solution. In order to obtained the result, sensitivity measurement was done by using the EGFET setup equipment with constant-current (100 µA) and constant-voltage (0.3 V) biasing interfacing circuit. TiO_2/ZnO bilayer film which the working electrode, act as the pH-sensitive membrane was connected to a commercial metal-oxide semiconductor FET (MOSFET). This MOSFET then was connected to the interfacing circuit. The sensitivity of the TiO2 thin film towards pH buffer solution was measured by dipping the sensing membrane in pH4, pH7 and pH10 buffer solution. These thin films were characterized by using Field Emission Scanning Electron Microscope (FESEM) to obtain the surface morphology of the composite bilayer films. In addition, I-V measurement was done in order to determine the electrical properties of the bilayer films. According to the result obtained in this experiment, bilayer film that spin at 4000 rpm, gave highest sensitivity which is 52.1 mV/pH. Relating the I-V characteristic of the thin films and sensitivity, the sensing membrane with higher conductivity gave better sensitivity.
Atomic force microscopy on domains in biological model membranes

NARCIS (Netherlands)

Rinia, H.A.

2001-01-01

This thesis describes the preparation and imaging of supported lipid bilayers, which can be regarded as biological modelmembranes, in the light of the formation of domains. The bilayers were prepared with either the Langmuir-Blodgett method, or with vesicle fusion. They were imaged with Atomic Force
A new combination of membranes and membrane reactors for improved tritium management in breeder blanket of fusion machines

International Nuclear Information System (INIS)

Demange, D.; Staemmler, S.; Kind, M.

2011-01-01

Tritium used as fuel in future fusion machines will be produced within the breeder blanket. The tritium extraction system recovers the tritium to be routed into the inner-fuel cycle of the machine. Accurate and precise tritium accountancy between both systems is mandatory to ensure a reliable operation. Handling in the blanket huge helium flow rates containing tritium as traces in molecular and oxide forms is challenging both for the process and the accountancy. Alternative tritium processes based on combinations of membranes and membrane reactors are proposed to facilitate the tritium management. The PERMCAT process is based on counter-current isotope swamping in a palladium membrane reactor. It allows recovering tritium efficiently from any chemical species. It produces a pure hydrogen stream enriched in tritium of advantage for integration upstream of the accountancy stage. A pre-separation and pre-concentration stage using new zeolite membranes has been studied to optimize the whole process. Such a combination could improve the tritium processes and facilitate accountancy in DEMO.
Interaction of Melittin with Negatively Charged Lipid Bilayers Supported on Gold Electrodes

International Nuclear Information System (INIS)

Juhaniewicz, Joanna; Sek, Slawomir

2016-01-01

ABSTRACT: The interactions of melittin, a cationic antimicrobial peptide, with model lipid membranes consisting of negatively charged phospholipids: 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) were investigated using electrochemical techniques and atomic force microscopy. Lipid bilayers were deposited on gold electrodes using a combination of Langmuir-Blodgett and Langmuir-Schaefer methods and the resulting membranes established a barrier for electron transfer between the electrode and the redox probe in the solution. After exposure to melittin, the blocking properties of the membranes were monitored using cyclic voltammetry and electrochemical impedance spectroscopy. It was observed that after treatment with peptide, the charge transfer through lipid bilayer is initially strongly inhibited. However, after longer exposure to melittin, the structure of the lipid film becomes less compact and the electrode reactions are facilitated due to the presence of numerous defect sites exposing bare substrate. We have assumed that such behavior reflects initial adsorption of melittin on top of the membrane and its further insertion which leads to formation of the pores or partial micellization of the lipid film. AFM imaging revealed that the exposure to 10 μM melittin solution induces significant structural changes in DMPG and DMPS membranes. However, melittin seems to affect their organization in a different manner. DMPG film appears to be more susceptible to peptide action compared with DMPS bilayer. In the latter case, long-time exposure to melittin does not result in the rupture of the membrane but rather leads to formation of pore-like defects. This observation is explained in terms of different nanomechanical properties of DMPG and DMPS films and different barrier for the reorientation and insertion of the peptide molecules into the membranes.
SAXS investigations on lipid membranes under osmotic stress

Energy Technology Data Exchange (ETDEWEB)

Rubim, R.L.; Vieira, V.; Gerbelli, B.B.; Teixeira da Silva, E.R.; Oliveira, C.L.P.; Oliveira, E.A. [Universidade de Sao Paulo (USP), Sao Paulo, SP (Brazil)

2012-07-01

Full text: In this work we, experimentally, investigate the interactions between lipid bilayers. A structural characterization is performed by small angle x-ray scattering (SAXS) on multilamellar systems under known osmotic pressure. Changes in the composition of membranes can modify their mechanical properties and structural parameters, like the flexibility of these membranes, which plays a key role on the determination of the tridimensional organization of bilayers. The membranes are composed of soya lecithin, where the major component is DPPC (Dipalmitoylphosphatidylcholine), and fatty acids are incorporated to the membrane in different concentrations, in order to turn the membrane more fluid. The membranes are inserted in a solution of PVP [poly(vinyl-pyrrolidone) - 40000] and the polymer will apply an osmotic pressure on them. The osmotic pressure is controlled by preparing PVP solutions of desired composition and, as we know the concentration of polymer in solution, we can obtain the intensity of the osmotic pressure. SAXS experiments were done in order to determine the distance between the bilayer. From the position of the Bragg peaks, the lamellar periodicity (the thickness of the membranes plus their distance of separation) was determined. Using theoretical model for the form and structure factors we fitted those experimental data and determined the thickness of the membranes. The distance between the membranes was controlled by the osmotic pressure (P) applied to the membranes and, for a given pressure, we determine the distance between the bilayers (a) on equilibrium. The experimental curve P(a) is theoretically described by the different contributions from van der Waals, hydration and fluctuation forces. From the fitting of experimental curves, relevant parameters characterizing the strength of the different interactions are obtained, such as Hamaker and rigidity constant [2, 3]. We observe that the separation between the bilayers on equilibrium is
NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

Science.gov (United States)

Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

2007-08-15

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.
Interaction of abscisic acid with phospholipid membranes

International Nuclear Information System (INIS)

Stillwell, W.; Brengle, B.; Hester, P.; Wassall, S.T.

1989-01-01

The plant hormone abscisic acid (ABA) is shown, under certain conditions, to greatly enhance the permeability of phospholipid bilayer membranes to the nonelectrolyte erythritol (followed spectrophotometrically by osmotic swelling) and the anion carboxyfluorescein (followed by fluorescence). The hormone is ineffective with single- and mixed-component phosphatidylcholine membranes in the liquid-crystalline or gel states. In contrast, substantial ABA-induced permeability is measured for two-component membranes containing lipids with different polar head groups or containing phosphatidylcholines with different acyl chains at temperatures where gel and liquid-crystalline phases coexist. Despite the large ABA-induced enhancement in bilayer permeability, no evidence for a substantial change at the molecular level was seen in the membranes by magnetic resonance techniques. 13 C NMR spin-lattice relaxation times, T 1 , in sonicated unilamellar vesicles and ESR of spin-labeled fatty acids intercalated into membranes showed negligible effect on acyl chain order and dynamics within the bilayer, while 31 P NMR of sonicated unilamellar vesicles indicated negligible effect on molecular motion and conformation in the head-group region. The authors propose that, instead of causing a general nonspecific perturbation to the membrane, the hormone acts at membrane defects formed due to mismatch in molecular packing where two different head groups or acyl chain states interface. Increased membrane disruption by ABA at these points of membrane instability could then produce an enhancement in permeability
Supported lipid bilayers with controlled curvature via colloidal lithography

DEFF Research Database (Denmark)

Sundh, Maria; Manandhar, Michal; Svedhem, Sofia

2011-01-01

Supported lipid bilayers (SLBs) at surfaces provide a route to quantitatively study molecular interactions with and at lipid membranes via different surface-based analytical techniques. Here, a method to fabricate SLBs with controlled curvatures, in the nanometer regime over large areas, is prese...
Membrane-Assisted Growth of DNA Origami Nanostructure Arrays

Science.gov (United States)

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes. PMID:25734977
Membrane-assisted growth of DNA origami nanostructure arrays.

Science.gov (United States)

Kocabey, Samet; Kempter, Susanne; List, Jonathan; Xing, Yongzheng; Bae, Wooli; Schiffels, Daniel; Shih, William M; Simmel, Friedrich C; Liedl, Tim

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors--a three-layered rectangular block and a Y-shaped DNA structure--to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.
Alamethicin in lipid bilayers: combined use of X-ray scattering and MD simulations.

Science.gov (United States)

Pan, Jianjun; Tieleman, D Peter; Nagle, John F; Kucerka, Norbert; Tristram-Nagle, Stephanie

2009-06-01

We study fully hydrated bilayers of two di-monounsaturated phospholipids diC18:1PC (DOPC) and diC22:1PC with varying amounts of alamethicin (Alm). We combine the use of X-ray diffuse scattering and molecular dynamics simulations to determine the orientation of alamethicin in model lipids. Comparison of the experimental and simulated form factors shows that Alm helices are inserted transmembrane at high humidity and high concentrations, in agreement with earlier results. The X-ray scattering data and the MD simulations agree that membrane thickness changes very little up to 1/10 Alm/DOPC. In contrast, the X-ray data indicate that the thicker diC22:1PC membrane thins with added Alm, a total decrease in thickness of 4 A at 1/10 Alm/diC22:1PC. The different effect of Alm on the thickness changes of the two bilayers is consistent with Alm having a hydrophobic thickness close to the hydrophobic thickness of 27 A for DOPC; Alm is then mismatched with the 7 A thicker diC22:1PC bilayer. The X-ray data indicate that Alm decreases the bending modulus (K(C)) by a factor of approximately 2 in DOPC and a factor of approximately 10 in diC22:1PC membranes (P/L approximately 1/10). The van der Waals and fluctuational interactions between bilayers are also evaluated through determination of the anisotropic B compressibility modulus.
Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

Science.gov (United States)

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-04-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. © 2015 Arlt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Model for the structure of the lipid bilayer

International Nuclear Information System (INIS)

Pastor, R.W.; Venable, R.M.; Karplus, M.

1991-01-01

A detailed model for the structure and dynamics of the interior of the lipid bilayer in the liquid crystal phase is presented. The model includes two classes of motion: (i) the internal dynamics of the chains, determined from Brownian dynamics simulations with a continuous version of the Marcelja mean-field potential, and (ii) noncollective reorientation (axial rotation and wobble) of the entire molecule, introduced by a cone model. The basic unit of the model is a single lipid chain with field parameters adjusted to fit the 2H order parameters and the frequency-dependent 13C NMR T1 relaxation times of dipalmitoyl phosphatidylcholine bilayers. The chain configurations obtained from the trajectory are used to construct a representation of the bilayer. The resulting lipid assembly is consistent with NMR, neutron diffraction, surface area, and density data. It indicates that a high degree of chain disorder and entanglement exists in biological membranes
Texture of lipid bilayer domains

DEFF Research Database (Denmark)

Jensen, Uffe Bernchou; Brewer, Jonathan R.; Midtiby, Henrik Skov

2009-01-01

We investigate the texture of gel (g) domains in binary lipid membranes composed of the phospholipids DPPC and DOPC. Lateral organization of lipid bilayer membranes is a topic of fundamental and biological importance. Whereas questions related to size and composition of fluid membrane domain...... are well studied, the possibility of texture in gel domains has so far not been examined. When using polarized light for two-photon excitation of the fluorescent lipid probe Laurdan, the emission intensity is highly sensitive to the angle between the polarization and the tilt orientation of lipid acyl...... chains. By imaging the intensity variations as a function of the polarization angle, we map the lateral variations of the lipid tilt within domains. Results reveal that gel domains are composed of subdomains with different lipid tilt directions. We have applied a Fourier decomposition method...
Characteristics of TiO{sub 2}/ZnO bilayer film towards pH sensitivity prepared by different spin coating deposition process

Energy Technology Data Exchange (ETDEWEB)

Rahman, Rohanieza Abdul, E-mail: rohanieza.abdrahman@gmail.com; Zulkefle, Muhammad Al Hadi, E-mail: alhadizulkefle@gmail.com [NANO-Electronic Centre, Faculty of Electrical Engineering, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor (Malaysia); Abdullah, Wan Fazlida Hanim, E-mail: wanfaz@salam.uitm.edu.my [Faculty of Electrical Engineering, Universiti Teknologi MARA (UiTM) Shah Alam, 40450 Shah Alam, Selangor (Malaysia); Rusop, M., E-mail: rusop@salam.uitm.com [NANO-Electronic Centre, Faculty of Electrical Engineering, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor (Malaysia); NANO-Science Technology Centre (NST), Institute of Science (IOS), Faculty of Applied Science, Universiti Teknologi MARA - UiTM, 40450 Shah Alam, Selangor (Malaysia); Herman, Sukreen Hana, E-mail: hana1617@salam.uitm.edu.my [NANO-Electronic Centre, Faculty of Electrical Engineering, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor (Malaysia); CoRe of Frontier Materials & Industry Applications, Faculty of Electrical Engineering, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor (Malaysia)

2016-07-06

In this study, titanium dioxide (TiO{sub 2}) and zinc oxide (ZnO) bilayer film for pH sensing application will be presented. TiO{sub 2}/ZnO bilayer film with different speed of spin-coating process was deposited on Indium Tin Oxide (ITO), prepared by sol-gel method. This fabricated bilayer film was used as sensing membrane for Extended Gate Field-Effect Transistor (EGFET) for pH sensing application. Experimental results indicated that the sensor is able to detect the sensitivity towards pH buffer solution. In order to obtained the result, sensitivity measurement was done by using the EGFET setup equipment with constant-current (100 µA) and constant-voltage (0.3 V) biasing interfacing circuit. TiO{sub 2}/ZnO bilayer film which the working electrode, act as the pH-sensitive membrane was connected to a commercial metal-oxide semiconductor FET (MOSFET). This MOSFET then was connected to the interfacing circuit. The sensitivity of the TiO2 thin film towards pH buffer solution was measured by dipping the sensing membrane in pH4, pH7 and pH10 buffer solution. These thin films were characterized by using Field Emission Scanning Electron Microscope (FESEM) to obtain the surface morphology of the composite bilayer films. In addition, I-V measurement was done in order to determine the electrical properties of the bilayer films. According to the result obtained in this experiment, bilayer film that spin at 4000 rpm, gave highest sensitivity which is 52.1 mV/pH. Relating the I-V characteristic of the thin films and sensitivity, the sensing membrane with higher conductivity gave better sensitivity.
Mesoscopic models of biological membranes

DEFF Research Database (Denmark)

Venturoli, M.; Sperotto, Maria Maddalena; Kranenburg, M.

2006-01-01

Phospholipids are the main components of biological membranes and dissolved in water these molecules self-assemble into closed structures, of which bilayers are the most relevant from a biological point of view. Lipid bilayers are often used, both in experimental and by theoretical investigations...... to coarse grain a biological membrane. The conclusion of this comparison is that there can be many valid different strategies, but that the results obtained by the various mesoscopic models are surprisingly consistent. A second objective of this review is to illustrate how mesoscopic models can be used...
Structure and hydration of membranes embedded with voltage-sensing domains.

Science.gov (United States)

Krepkiy, Dmitriy; Mihailescu, Mihaela; Freites, J Alfredo; Schow, Eric V; Worcester, David L; Gawrisch, Klaus; Tobias, Douglas J; White, Stephen H; Swartz, Kenton J

2009-11-26

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

Directory of Open Access Journals (Sweden)

Yujie Zhu

2015-08-01

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

2016-05-01

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

Science.gov (United States)

Weinreb, Gabriel; Lentz, Barry R

2007-06-01

We propose a model that accounts for the time courses of PEG-induced fusion of membrane vesicles of varying lipid compositions and sizes. The model assumes that fusion proceeds from an initial, aggregated vesicle state ((A) membrane contact) through two sequential intermediate states (I(1) and I(2)) and then on to a fusion pore state (FP). Using this model, we interpreted data on the fusion of seven different vesicle systems. We found that the initial aggregated state involved no lipid or content mixing but did produce leakage. The final state (FP) was not leaky. Lipid mixing normally dominated the first intermediate state (I(1)), but content mixing signal was also observed in this state for most systems. The second intermediate state (I(2)) exhibited both lipid and content mixing signals and leakage, and was sometimes the only leaky state. In some systems, the first and second intermediates were indistinguishable and converted directly to the FP state. Having also tested a parallel, two-intermediate model subject to different assumptions about the nature of the intermediates, we conclude that a sequential, two-intermediate model is the simplest model sufficient to describe PEG-mediated fusion in all vesicle systems studied. We conclude as well that a fusion intermediate "state" should not be thought of as a fixed structure (e.g., "stalk" or "transmembrane contact") of uniform properties. Rather, a fusion "state" describes an ensemble of similar structures that can have different mechanical properties. Thus, a "state" can have varying probabilities of having a given functional property such as content mixing, lipid mixing, or leakage. Our data show that the content mixing signal may occur through two processes, one correlated and one not correlated with leakage. Finally, we consider the implications of our results in terms of the "modified stalk" hypothesis for the mechanism of lipid pore formation. We conclude that our results not only support this hypothesis but
Molybdenum-gold proximity bilayers as transition edge sensors for microcalorimeters and bolometers

International Nuclear Information System (INIS)

Chen, T.C.; Bier, A.; DiCamillo, B.; Finkbeiner, F. M.

1999-01-01

Mo/Au proximity bilayers as transition edge sensors (TESs) are promising candidates for low-temperature thermometry. The transition temperature of the bilayers can be easily tuned between 50 and 600 mK, yielding sensors which can be used in a variety of calorimetric and bolometric applications. With phase transition widths of less than 1 mK, Mo/Au TESs show very high temperature sensitivity (d(logR)/d(logT)∼2500). Also, Mo/Au TESs show improved thermal and chemical stability compared to most other bilayer configurations. Fabrication issues and detector performance of Mo/Au TESs on Si 3 N 4 membranes are discussed. (author)
SANS study of the unilamellar DMPC vesicles. The fluctuation model of lipid bilayer

International Nuclear Information System (INIS)

Kiselev, M.A.; Zemlyanaya, E.V.; Vinod, A.

2003-01-01

On the basis of the separated form-factors model, parameters of the polydispersed unilamellar DMPC vesicle population are analyzed. The neutron scattering length density across the membrane is simulated on the basis of fluctuated model of lipid bilayer. The hydration of vesicle is described by sigmoid distribution function of the water molecules. The results of fitting of the experimental data obtained at the small angle spectrometer SANS-I, PSI (Switzerland) are: average vesicle radius 272±0.4 Armstrong, polydispersity of the radius 27 %, membrane thickness 50.6± Armstrong, thickness of hydrocarbon chain region 21.4±2.8 Armstrong, number of water molecules located per lipid molecule 13±1, and DMPC surface area 59±2 Armstrong 2 . The calculated water distribution function across the bilayer directly explains why lipid membrane is easy penetrated by water molecules
The effects of ethylene oxide containing lipopolymers and tri-block copolymers on lipid bilayers of dipalmitoylphosphatidylcholine

DEFF Research Database (Denmark)

Baekmark, T. R.; Pedersen, S.; Jorgensen, K.

1997-01-01

oxide moity, anchored to the bilayer by a 1,2-dioctadecanoyl-s,n-glycero-3-phosphoethanolamine (DC18PE) lipid. The second type, which is a novel type of membrane-spanning object, is an amphiphilic tri-block copolymer composed of two hydrophilic stretches of polyethylene oxide separated by a hydrophobic...... stretch of polystyrene. Hence the tri-block copolymer may act as a membrane-spanning macromolecule mimicking an amphiphilic protein or polypeptide. Differential scanning calorimetry is used to determine a partial phase diagram for the lipopolymer systems and to assess the amount of lipopolymer that can...... be loaded into DC16PC lipid bilayers before micellization takes place. Unilamellar and micellar phase structures are investigated by fluorescence quenching using bilayer permeating dithionite. The chain length-dependent critical lipopolymer concentration, denoting the lamellar-to-micellar phase transition...
Membrane bridging and hemifusion by denaturated Munc18.

Directory of Open Access Journals (Sweden)

Yi Xu

Full Text Available Neuronal Munc18-1 and members of the Sec1/Munc18 (SM protein family play a critical function(s in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1 was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37 °C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate.
On the application of the MARTINI coarse-grained model to immersion of a protein in a phospholipid bilayer

Energy Technology Data Exchange (ETDEWEB)

Mustafa, Ghulam, E-mail: Ghulam.Mustafa@h-its.org, E-mail: rebecca.wade@h-its.org; Nandekar, Prajwal P.; Yu, Xiaofeng [Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), Schloß-Wolfsbrunnenweg 35, 69118 Heidelberg (Germany); Wade, Rebecca C., E-mail: Ghulam.Mustafa@h-its.org, E-mail: rebecca.wade@h-its.org [Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), Schloß-Wolfsbrunnenweg 35, 69118 Heidelberg (Germany); Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, INF 282, 69120 Heidelberg (Germany); Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, INF 368, 69120 Heidelberg (Germany)

2015-12-28

An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions.
On the application of the MARTINI coarse-grained model to immersion of a protein in a phospholipid bilayer

International Nuclear Information System (INIS)

Mustafa, Ghulam; Nandekar, Prajwal P.; Yu, Xiaofeng; Wade, Rebecca C.

2015-01-01

An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions
Dynamics, Surface Electrostatics and Phase Properties of Nanoscale Curved Lipid Bilayers

Science.gov (United States)

Koolivand, Amir

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

NARCIS (Netherlands)

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

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand
The Physical Properties of Ceramides in Membranes.

Science.gov (United States)

Alonso, Alicia; Goñi, Félix M

2018-05-20

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

International Nuclear Information System (INIS)

Solovjov, D.V.; Gordelyij, V.Yi.; Gorshkova, Yu.Je.; Yivan'kov, O.Yi.; Koval'ov, Yu.S.; Kuklyin, A.Yi.; Solovjov, D.V.; Bulavyin, L.A.; Yivan'kov, O.Yi.; Nyikolajenko, T.Yu.; Kuklyin, A.Yi.; Gordelyij, V.Yi.; Gordelyij, V.Yi.

2012-01-01

Lipid bilayer structure investigation results obtained with small angle neutron scattering method at the Joint Institute for Nuclear Research IBR-2M nuclear reactor (Dubna, Russia) are presented. Experiment has been performed with small angle neutron scattering spectrometer YuMO, upgraded with the apparatus for performing PV-T measurements on the substance under investigation. D 2 O-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liquid system, presenting the model of natural live membrane, has been taken as the sample for investigations. The lipid bilayer spatial period was measured in experiment along with isothermal compressibility simultaneously at different pressures. It has been shown, that the bilayer structural transition from ripple (wavelike gel-phase) phase to liquid-crystal phase is accompanied with anomalous rise of isothermal compressibility, indicating occurrence of the phase transition.
Optical response of Al/Ti bilayer transition edge sensors

International Nuclear Information System (INIS)

Zhang Qing-Ya; Liu Jian-She; Dong Wen-Hui; He Gen-Fang; Li Tie-Fu; Chen Wei; Wang Tian-Shun; Zhou Xing-Xiang

2014-01-01

We report the optical response characteristics of Al/Ti bilayer transition edge sensors (TESs), which are mainly comprised of Al/Ti bilayer thermometers and suspended SiN membranes for thermal isolation. The measurement was performed in a 3 He sorption refrigerator and the device's response to optical pulses was investigated using a pulsed laser source. Based on these measurements, we obtained the effective recovery time (τ eff ) of the devices at different biases and discussed the dependence of τ eff on the bias. The device with a 940 μm × 940 μm continuous suspended SiN membrane demonstrated a fast response speed with τ eff = 3.9 μs, which indicates a high temperature sensitivity (α = T/R · dR/dT = 326). The results also showed that the TES exhibits good linearity under optical pulses of variable widths. (interdisciplinary physics and related areas of science and technology)
Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode

OpenAIRE

Haag, Lars; Garoff, Henrik; Xing, Li; Hammar, Lena; Kan, Sin-Tau; Cheng, R.Holland

2002-01-01

In the icosahedral (T = 4) Semliki Forest virus, the envelope protomers, i.e. E1–E2 heterodimers, make one-to-one interactions with capsid proteins below the viral lipid bilayer, transverse the membrane and form an external glycoprotein shell with projections. The shell is organized by protomer domains interacting as hexamers and pentamers around shell openings at icosahedral 2- and 5-fold axes, respectively, and the projections by other domains associating as trimers at 3- and quasi 3-fold a...
NMR structural studies of peptides and proteins in membranes

Energy Technology Data Exchange (ETDEWEB)

Opella, S J [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry

1994-12-31

The use of NMR methodology in structural studies is described as applicable to larger proteins, considering that the majority of membrane proteins is constructed from a limited repertoire of structural and dynamic elements. The membrane associated domains of these proteins are made up of long hydrophobic membrane spanning helices, shorter amphipathic bridging helices in the plane of the bilayer, connecting loops with varying degrees of mobility, and mobile N- and C- terminal sections. NMR studies have been successful in identifying all of these elements and their orientations relative to each other and the membrane bilayer 19 refs., 9 figs.
1,2-Dielaidoylphosphocholine/1,2-dimyristoylphosphoglycerol supported phospholipid bilayer formation in calcium and calcium-free buffer

International Nuclear Information System (INIS)

Evans, Kervin O.

2012-01-01

Phospholipid membranes are useful in the field of biocatalysis because a supported phospholipid membrane can create a biomimetic platform where biocatalytic processes can readily occur. In this work, supported bilayer formation from sonicated phospholipid vesicles containing 1,2-dielaidoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] was studied using a quartz crystal microbalance with dissipation monitoring and an atomic force microscope. The molar percentages of DEPC and DMPG were varied to determine the effect of overall lipid composition on supported bilayer formation. This work also explored the effect that calcium ion concentration had on supported bilayer formation. Results show that vesicles with up to 50 mol% dimyristoylphosphoglycerol can form a supported bilayer without the presence of calcium ions; however, supported bilayer formation in calcium buffer was inhibited as the anionic (negatively charged) lipid concentration increased. Data suggest that supported phospholipid bilayer formation in the absence of Ca 2+ from vesicles containing negatively charged lipids is specific to phosphatidylglycerol. - Highlights: ► SPB formation of DEPC vesicles containing 0 to 50 mol% DMPG monitored using QCM-D. ► Ca 2+ inhibited SPB formation of DEPC vesicles containing 30 to 50 mol% DMPG. ► Vesicles containing DMPG at 0 to 50 mol% formed SPB in buffer free of Ca 2+ .
Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

OpenAIRE

Notman, Rebecca; Anwar, Jamshed; Briels, W. J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel pha...
Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

Science.gov (United States)

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

2015-04-01

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

Science.gov (United States)

Olmsted, Peter; Das, Chinmay; Noro, Massimo

2012-02-01

The stratum corneum is the outermost layer of skin, and serves as a protective barrier against external agents, and to control moisture. It comprises keratin bodies (corneocytes) embedded in a matrix of lipid bilayers. Unlike the more widely studied phospholipid bilayers, the SC bilayers are typically in a gel-like state. Moreover, the SC membrane composition is radically different from more fluid counterparts: it comprises single tailed fatty acids, ceramides, and cholesterol; with many distinct ceramides possessing different lengths of tails, and always with two tails of different lengths. I will present insight from computer simulations into the morphology, mechanical properties, and diffusion (barrier) properties of these highly heterogeneous membranes. Our results provide some clue as to the design principles for the SC membrane, and is an excellent example of the use of wide polydispersity by natural systems.
Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

Science.gov (United States)

Doxastakis, Manolis; Prakash, Anupam; Janosi, Lorant

2011-03-01

Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces.

Diffusion studies on permeable nitroxyl spin probes through bilayer lipid membranes: A low frequency ESR study

International Nuclear Information System (INIS)

Meenakumari, V.; Benial, A. Milton Franklin; Utsumi, Hideo; Ichikawa, Kazuhiro; Yamada, Ken-ichi; Hyodo, Fuminori; Jawahar, A.

2015-01-01

Electron spin resonance (ESR) studies were carried out for permeable 2mM 14 N-labeled deutrated 3 Methoxy carbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) in pure water and 1mM, 2mM, 3mM, 4mM concentration of 14N-labeled deutrated MC-PROXYL in 400mM concentration of liposomal solution by using a 300 MHz ESR spectrometer. The ESR parameters such as linewidth, hyperfine coupling constant, g-factor, partition parameter and permeability were reported for these samples. The line broadening was observed for the nitroxyl spin probe in the liposomal solution. The line broadening indicates that the high viscous nature of the liposomal solution. The partition parameter and permeability values indicate the maximum diffusion of nitroxyl spin probes in the bilayer lipid membranes at 2 mM concentration of nitroxyl radical. This study illustrates that ESR can be used to differentiate between the intra and extra- membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe. From the ESR results, the spin probe concentration was optimized as 2mM in liposomal solution for ESR phantom studies/imaging, invivo and invitro experiments
The interaction of new piroxicam analogues with lipid bilayers--a calorimetric and fluorescence spectroscopic study.

Science.gov (United States)

Maniewska, Jadwiga; Szczęśniak-Sięga, Berenika; Poła, Andrzej; Sroda-Pomianek, Kamila; Malinka, Wiesław; Michalak, Krystyna

2014-01-01

The purpose of the present paper was to assess the ability of new piroxicam analogues to interact with the lipid bilayers. The results of calorimetric and fluorescence spectroscopic experiments of two new synthesized analogues of piroxicam, named PR17 and PR18 on the phase behavior of phospholipid bilayers and fluorescence quenching of fluorescent probes (Laurdan and Prodan), which molecular location within membranes is known with certainty, are shown in present work. The presented results revealed that, depending on the details of chemical structure, the studied compounds penetrated the lipid bilayers.
Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION

NARCIS (Netherlands)

Ansari, Israr-Ul H.; Longacre, Melissa J.; Paulusma, Coen C.; Stoker, Scott W.; Kendrick, Mindy A.; MacDonald, Michael J.

2015-01-01

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote
Experimental Investigations of Direct and Converse Flexoelectric Effect in Bilayer Lipid Membranes.

Science.gov (United States)

Todorov, Angelio Todorov

Flexoelectric coefficients (direct and converse), electric properties (capacitance and resistivity) and mechanical properties (thickness and elastic coefficients) have been determined for bilayer lipid membranes (BLMs) prepared from egg yolk lecithin (EYL), glycerol monoleate (GMO), phosphatidyl choline (PC) and phosphatidyl serine (PS) as a function of frequency, pH and surface charge modifiers. Direct flexoelectric effect manifested itself in the development of microvolt range a.c. potential (U_{f}) upon subjecting one side of a BLM to an oscillating hydrostatic pressure, in the 100-1000 Hz range. Operationally, the flexoelectric coefficient (f) is expressed by the ratio between U_{f} and the change of curvature (c) which accompanied the flexing of the membrane. Membrane curvature was determined by means of either the electric method (capacitance microphone effect) or by the newly developed method of stroboscopic interferometry. Real-time stroboscopic interferometry coupled with simultaneous electric measurements, provided a direct method for the determination of f. Two different frequency regimes of f were recognized. At low frequencies (300 Hz), associated with free mobility of the surfactant, f-values of 24.1 times 10^{-19} and 0.87 times 10^ {-19} Coulombs were obtained for PC and GMO BLMs. At high frequencies (>300 Hz), associated with blocked mobility of the surfactant, f-values of 16.5 times 10^ {-19} and 0.30 times 10^{-19} Coulombs were obtained for PC and GMO BLMs. The theoretically calculated value for the GMO BLM oscillating at high frequency (0.12 times 10^{-19 } Coulombs) agreed well with that determined experimentally (0.3 times 10 ^{-19} Coulombs). For charged bovine brain PS BLM the observed flexocoefficient was f = 4.0 times 10^{ -18} Coulombs. Converse flexoelectric effect manifested itself in voltage-induced BLM curvature. Observations were carried out on uranyl acetate (UA) stabilized PS BLM under a.c. excitation. Frequency dependence of f
Calculation of the electrostatic potential of lipid bilayers from molecular dynamics simulations: methodological issues

DEFF Research Database (Denmark)

Gurtovenko, Andrey A; Vattulainen, Ilpo

2009-01-01

of the electrostatic potential from atomic-scale molecular dynamics simulations of lipid bilayers. We discuss two slightly different forms of Poisson equation that are normally used to calculate the membrane potential: (i) a classical form when the potential and the electric field are chosen to be zero on one...... systems). For symmetric bilayers we demonstrate that both approaches give essentially the same potential profiles, provided that simulations are long enough (a production run of at least 100 ns is required) and that fluctuations of the center of mass of a bilayer are properly accounted for. In contrast...
Biomimetic Membrane Arrays on Cast Hydrogel Supports

DEFF Research Database (Denmark)

Roerdink-Lander, Monique; Ibragimova, Sania; Rein Hansen, Christian

2011-01-01

, provides mechanical support but at the cost of small molecule transport through the membrane−support sandwich. To stabilize biomimetic membranes while allowing transport through a membrane−support sandwich, we have investigated the feasibility of using an ethylene tetrafluoroethylene (ETFE......)/hydrogel sandwich as the support. The sandwich is realized as a perforated surface-treated ETFE film onto which a hydrogel composite support structure is cast. We report a simple method to prepare arrays of lipid bilayer membranes with low intrinsic electrical conductance on the highly permeable, self......-supporting ETFE/hydrogel sandwiches. We demonstrate how the ETFE/hydrogel sandwich support promotes rapid self-thinning of lipid bilayers suitable for hosting membrane-spanning proteins....
Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

Science.gov (United States)

Endo, Masayuki; Sugiyama, Hiroshi

2015-01-01

Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed ‘lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space. PMID:26310995
Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

Science.gov (United States)

Suzuki, Yuki; Endo, Masayuki; Sugiyama, Hiroshi

2015-08-01

Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed `lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space.
The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

International Nuclear Information System (INIS)

Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.; Santos, Nuno C.

2010-01-01

Research highlights: → Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. → Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. → This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.
In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence

Science.gov (United States)

Figueira, T. N.; Palermo, L. M.; Veiga, A. S.; Huey, D.; Alabi, C. A.; Santos, N. C.; Welsch, J. C.; Mathieu, C.; Niewiesk, S.; Moscona, A.

2016-01-01

ABSTRACT Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides. PMID:27733647
Structure and properties of cell membranes. Volume 3: Methodology and properties of membranes

International Nuclear Information System (INIS)

Benga, G.

1985-01-01

This book covers the topics: Quantum chemical approach to study the mechanisms of proton translocation across membranes through protein molecules; monomolecular films as biomembrane models; planar lipid bilayers in relation to biomembranes; relation of liposomes to cell membranes; reconstitution of membrane transport systems; structure-function relationships in cell membranes as revealed by X-ray techniques; structure-function relationships in cell membranes as revealed by spin labeling ESR; structure and dynamics of cell membranes as revealed by NMR techniques; the effect of dietary lipids on the composition and properties of biological membranes and index
CURVATURE-DRIVEN MOLECULAR FLOW ON MEMBRANE SURFACE.

Science.gov (United States)

Mikucki, Michael; Zhou, Y C

2017-01-01

This work presents a mathematical model for the localization of multiple species of diffusion molecules on membrane surfaces. Morphological change of bilayer membrane in vivo is generally modulated by proteins. Most of these modulations are associated with the localization of related proteins in the crowded lipid environments. We start with the energetic description of the distributions of molecules on curved membrane surface, and define the spontaneous curvature of bilayer membrane as a function of the molecule concentrations on membrane surfaces. A drift-diffusion equation governs the gradient flow of the surface molecule concentrations. We recast the energetic formulation and the related governing equations by using an Eulerian phase field description to define membrane morphology. Computational simulations with the proposed mathematical model and related numerical techniques predict (i) the molecular localization on static membrane surfaces at locations with preferred mean curvatures, and (ii) the generation of preferred mean curvature which in turn drives the molecular localization.
Neutron scattering investigations of the lipid bilayer structure pressure dependence

Directory of Open Access Journals (Sweden)

D. V. Soloviov

2012-03-01

Full Text Available Lipid bilayer structure investigation results obtained with small angle neutron scattering method at the Joint Institute for Nuclear Research IBR-2M nuclear reactor (Dubna, Russia are presented. Experiment has been per-formed with small angle neutron scattering spectrometer YuMO, upgraded with the apparatus for performing P-V-T measurements on the substance under investigation. D2O-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC liquid system, presenting the model of natural live membrane, has been taken as the sample for investiga-tions. The lipid bilayer spatial period was measured in experiment along with isothermal compressibility simulta-neously at different pressures. It has been shown, that the bilayer structural transition from ripple (wavelike gel-phase phase to liquid-crystal phase is accompanied with anomalous rise of isothermal compressibility, indicat-ing occurrence of the phase transition.
The impact of resveratrol in lipid bilayers

DEFF Research Database (Denmark)

Shen, Chen; de Ghellinck, Alexis; Fragneto, Giovanna

The natural antioxidant resveratrol, contained in the skin of red grape and accordingly in their wines, is hold liable for health impacts such as cardiovascular protection and anti-oxidative effect. Clinical trials of resveratrol as prophylactic or even therapeutic drug are ongoing. However, basic...... knowledge on its probable working mechanism is rare. In this biophysical study, neutron reflectometry was used to investigate the direct impact of resveratrol on lipid membranes with solid supported bilayers. When interacting with di- palmitoyl-phosphatidyl-choline (DPPC) bilayers, resveratrol accumulates...... in between the headgroups but is absent in the hydrophobic core. Without a biogenic removal mechanism, the headgroup region may host up to ~25 mol% of resveratrol. The average thickness and the interfacial roughness of the headgroup layer are increased. From the structural results, the average tilting...
Modulated phases of phospholipid bilayers induced by tocopherols.

Science.gov (United States)

Kamal, Md Arif; Raghunathan, V A

2012-11-01

The influence of α-, γ- and δ-tocopherols on the structure and phase behavior of dipalmitoyl phosphatidylcholine (DPPC) bilayers has been determined from X-ray diffraction studies on oriented multilayers. In all the three cases the main-transition temperature (T(m)) of DPPC was found to decrease with increasing tocopherol concentration up to around 25 mol%. Beyond this the main transition is suppressed in the case of γ-tocopherol, whereas T(m) becomes insensitive to composition in the other two cases. The pre-transition is found to be suppressed over a narrow tocopherol concentration range between 7.5 and 10 mol% in DPPC-γ-tocopherol and DPPC-δ-tocopherol bilayers, and the ripple phase occurs down to the lowest temperature studied. In all the three cases a modulated phase is observed above a tocopherol concentration of about 10 mol%, which is similar to the P(β) phase reported in DPPC-cholesterol bilayers. This phase is found to occur even in excess water conditions at lower tocopherol concentrations, and consists of bilayers with periodic height modulation. These results indicate the ability of tocopherols to induce local curvature in membranes, which could be important for some of their biological functions. Copyright © 2012 Elsevier B.V. All rights reserved.
'Boomerang'-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR.

Science.gov (United States)

Afonin, Sergii; Dürr, Ulrich H N; Glaser, Ralf W; Ulrich, Anne S

2004-02-01

Solid state (19)F NMR revealed the conformation and alignment of the fusogenic peptide sequence B18 from the sea urchin fertilization protein bindin embedded in flat phospholipid bilayers. Single (19)F labels were introduced into nine distinct positions along the wild-type sequence by substituting each hydrophobic amino acid, one by one, with L-4-fluorophenylglycine. Their anisotropic chemical shifts were measured in uniaxially oriented membrane samples and used as orientational constraints to model the peptide structure in the membrane-bound state. Previous (1)H NMR studies of B18 in 30% TFE and in detergent micelles had shown that the peptide structure consists of two alpha-helical segments that are connected by a flexible hinge. This helix-break-helix motif was confirmed here by the solid-state (19)F NMR data, while no other secondary structure (beta-sheet, 3(10)-helix) was compatible with the set of orientational constraints. For both alpha-helical segments we found that the helical conformation extends all the way to the respective N- and C-termini of the peptide. Analysis of the corresponding tilt and azimuthal rotation angles showed that the N-terminal helix of B18 is immersed obliquely into the bilayer (at a tilt angle tau approximately 54 degrees), whereas the C-terminus is peripherally aligned (tau approximately 91 degrees). The azimuthal orientation of the two segments is consistent with the amphiphilic distribution of side-chains. The observed 'boomerang'-like mode of insertion into the membrane may thus explain how peptide binding leads to lipid dehydration and acyl chain perturbation as a prerequisite for bilayer fusion to occur. Copyright 2004 John Wiley & Sons, Ltd.
Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

International Nuclear Information System (INIS)

Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

2008-01-01

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxΦ domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1 NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of
Primordial membranes

DEFF Research Database (Denmark)

Hanczyc, Martin M; Monnard, Pierre-Alain

2017-01-01

Cellular membranes, which are self-assembled bilayer structures mainly composed of lipids, proteins and conjugated polysaccharides, are the defining feature of cell physiology. It is likely that the complexity of contemporary cells was preceded by simpler chemical systems or protocells during...... the various evolutionary stages that led from inanimate to living matter. It is also likely that primitive membranes played a similar role in protocell 'physiology'. The composition of such ancestral membranes has been proposed as mixtures of single hydrocarbon chain amphiphiles, which are simpler versions...
Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites.

Science.gov (United States)

Yu, Haijia; Liu, Yinghui; Gulbranson, Daniel R; Paine, Alex; Rathore, Shailendra S; Shen, Jingshi

2016-04-19

Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation.
Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

Energy Technology Data Exchange (ETDEWEB)

Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu (Univ. of Tokyo (Japan))

1990-01-09

Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

3D pressure field in lipid membranes and membrane-protein complexes

DEFF Research Database (Denmark)

Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti

2009-01-01

We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...... a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane....
Electrostatic control of the dynamics of lipid bilayer self-spreading using a nanogap gate

International Nuclear Information System (INIS)

Kashimura, Y; Sumitomo, K; Furukawa, K

2014-01-01

The electrostatic control of lipid bilayer self-spreading was investigated using a device equipped with a nanogap gate. A series of mixtures containing negatively charged and uncharged lipids were employed to tune the charge of a membrane. We found that when a voltage is applied on a lipid bilayer passing through a nanogap, the effect of a voltage application on the dynamics depended largely on the charge of the membrane. For rich charged lipid compositions (>10 mol%), the self-spreading was electrostatically controlled applying an electric field to the nanogap. The origin of the behaviour is the electrostatic trapping of charged lipids. The trapped lipids close the nanogap gate, thus preventing any lipid molecules from passing through it. For poor charged lipid compositions (∼1 mol%), no electrostatic trapping occurred even when a lipid bilayer reached the nanogap. Instead, we observed the cessation of self-spreading after a sufficient post-passage time interval, indicating that the translational flow force of self-spreading overcomes the trapping force. For uncharged lipid compositions, there was no electrostatic trapping throughout the measurement. The results suggest that the lipid charge plays a vital role in the electrostatic control mechanism and allow us to control lipid bilayer formation both spatially and temporally. (paper)
Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

Science.gov (United States)

Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

2015-07-01

Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.
Bursting Bubbles and Bilayers

Directory of Open Access Journals (Sweden)

Steven P. Wrenn, Stephen M. Dicker, Eleanor F. Small, Nily R. Dan, Michał Mleczko, Georg Schmitz, Peter A. Lewin

2012-01-01

Full Text Available This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol (PEG - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented
Layer configurations comparison of bilayer-films for EGFET pH sensor application

Science.gov (United States)

Rahman, R. A.; Zulkefle, M. A.; Yusof, K. A.; Abdullah, W. F. H.; Rusop, M.; Herman, S. H.

2018-05-01

The comparison between bilayer configurations were presented in this paper. TiO2 and ZnO layer configurations were manipulated in order to investigate which configuration produce highest sensing performance to be applied as EGFET pH sensor. Both of the materials were deposited together as the bilayer film. The configurations were manipulated between TiO2/ZnO and ZnO/TiO2. ITO was used as the substrate in this study and both of the materials were deposited by using sol-gel spin coating technique. After deposition process, these bilayer film then undergone for EGFET pH sensor measurement and physical characterization. The EGFET pH sensor measurement was done by dipping the fabricated bilayer film into three different pH values, which is pH4, pH7 and pH10. Bilayer film act as the pH-sensitive membrane, which connected to the commercial metal-oxide semiconductor FET (MOSFET). This MOSFET was connected to the interfacing circuit. Voltage output obtained were recorded and the graph was plotted by using the data recorded. Based on the EGFET pH sensor measurement, TiO2/ZnO bilayer film exhibit higher sensing performance, compared with ZnO/TiO2. TiO2/ZnO bilayer film produced 53.10 mV/pH with the linearity value of 0.9913. Afterwards, fabricated bilayer films then were characterized with AFM to explore their surface roughness and surface topography behavior.
Interaction of PLGA and trimethyl chitosan modified PLGA nanoparticles with mixed anionic/zwitterionic phospholipid bilayers studied using molecular dynamics simulations

Science.gov (United States)

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

2012-02-01

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

International Nuclear Information System (INIS)

Yang, J.; Martí, J.; Calero, C.

2014-01-01

Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10 −5 cm 2 /s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10 −8 cm 2 /s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2–8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of its interaction
Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes

Energy Technology Data Exchange (ETDEWEB)

Yang, J.; Martí, J., E-mail: jordi.marti@upc.edu [Department of Physics and Nuclear Engineering, Technical University of Catalonia-Barcelona Tech, B4-B5 Northern Campus, Jordi Girona 1-3, 08034 Barcelona, Catalonia (Spain); Calero, C. [Department of Physics and Nuclear Engineering, Technical University of Catalonia-Barcelona Tech, B4-B5 Northern Campus, Jordi Girona 1-3, 08034 Barcelona, Catalonia (Spain); Center for Polymer Studies, Department of Physics, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215 (United States)

2014-03-14

Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10{sup −5} cm{sup 2}/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10{sup −8} cm{sup 2}/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2–8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of
Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

Science.gov (United States)

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

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

Science.gov (United States)

D'Angelo, S. M.; Camesano, T. A.; Nagarajan, R.

2011-12-01

The same properties that give nanoparticles their enhanced function, such as high surface area, small size, and better conductivity, can also alter the cytotoxicity of nanomaterials. Ultimately, many of these nanomaterials will be released into the environment, and can cause cytotoxic effects to environmental bacteria, aquatic organisms, and humans. Previous results from our laboratory suggest that nanoparticles can have a detrimental effect on cells, depending on nanoparticle size. It is our goal to characterize the properties of nanomaterials that can result in membrane destabilization. We tested the effects of nanoparticle size and chemical functionalization on nanoparticle-membrane interactions. Gold nanoparticles at 2, 5,10, and 80 nm were investigated, with a concentration of 1.1x1010 particles/mL. Model cell membranes were constructed of of L-α-phosphatidylcholine (egg PC), which has negatively charged lipid headgroups. A quartz crystal microbalance with dissipation (QCM-D) was used to measure frequency changes at different overtones, which were related to mass changes corresponding to nanoparticle interaction with the model membrane. In QCM-D, a lipid bilayer is constructed on a silicon dioxide crystal. The crystals, oscillate at different harmonic frequencies depending upon changes in mass or energy dissipation. When mass is added to the crystal surface, such as through addition of a lipid vesicle solution, the frequency change decreases. By monitoring the frequency and dissipation, we could verify that a supported lipid bilayer (SLB) formed on the silica surface. After formation of the SLB, the nanoparticles can be added to the system, and the changes in frequency and dissipation are monitored in order to build a mechanistic understanding of nanoparticle-cell membrane interactions. For all of the smaller nanoparticles (2, 5, and 10 nm), nanoparticle addition caused a loss of mass from the lipid bilayer, which appears to be due to the formation of holes
Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy

NARCIS (Netherlands)

Vermeer, J.E.M.; van Munster, E.B.; Vischer, N.O.; Gadella, T.

2004-01-01

Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region
pH sensing by lipids in membranes: The fundamentals of pH-driven migration, polarization and deformations of lipid bilayer assemblies.

Science.gov (United States)

Angelova, Miglena I; Bitbol, Anne-Florence; Seigneuret, Michel; Staneva, Galya; Kodama, Atsuji; Sakuma, Yuka; Kawakatsu, Toshihiro; Imai, Masayuki; Puff, Nicolas

2018-03-06

Most biological molecules contain acido-basic groups that modulate their structure and interactions. A consequence is that pH gradients, local heterogeneities and dynamic variations are used by cells and organisms to drive or regulate specific biological functions including energetic metabolism, vesicular traffic, migration and spatial patterning of tissues in development. While the direct or regulatory role of pH in protein function is well documented, the role of hydrogen and hydroxyl ions in modulating the properties of lipid assemblies such as bilayer membranes is only beginning to be understood. Here, we review approaches using artificial lipid vesicles that have been instrumental in providing an understanding of the influence of pH gradients and local variations on membrane vectorial motional processes: migration, membrane curvature effects promoting global or local deformations, crowding generation by segregative polarization processes. In the case of pH induced local deformations, an extensive theoretical framework is given and an application to a specific biological issue, namely the structure and stability of mitochondrial cristae, is described. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018 Elsevier B.V. All rights reserved.
The Influence of Cholesterol on Fast Dynamics Inside of Vesicle and Planar Phospholipid Bilayers Measured with 2D IR Spectroscopy.

Science.gov (United States)

Kel, Oksana; Tamimi, Amr; Fayer, Michael D

2015-07-23

Phospholipid bilayers are frequently used as models for cell membranes. Here the influence of cholesterol on the structural dynamics in the interior of 1,2-dilauroyl-sn-glycero-3-phosphocholine (dilauroylphosphatidylcholine, DLPC) vesicles and DLPC planar bilayers are investigated as a function of cholesterol concentration. 2D IR vibrational echo spectroscopy was performed on the antisymmetric CO stretch of the vibrational probe molecule tungsten hexacarbonyl, which is located in the interior alkyl regions of the bilayers. The 2D IR experiments measure spectral diffusion, which is caused by the structural fluctuations of the bilayers. The 2D IR measurements show that the bilayer interior alkyl region dynamics occur on time scales ranging from a few picoseconds to many tens of picoseconds. These are the time scales of the bilayers' structural dynamics, which act as the dynamic solvent bath for chemical processes of membrane biomolecules. The results suggest that at least a significant fraction of the dynamics arise from density fluctuations. Samples are studied in which the cholesterol concentration is varied from 0% to 40% in both the vesicles (72 nm diameter) and fully hydrated planar bilayers in the form of aligned multibilayers. At all cholesterol concentrations, the structural dynamics are faster in the curved vesicle bilayers than in the planar bilayers. As the cholesterol concentration is increased, at a certain concentration there is a sudden change in the dynamics, that is, the dynamics abruptly slow down. However, this change occurs at a lower concentration in the vesicles (between 10% and 15% cholesterol) than in the planar bilayers (between 25% and 30% cholesterol). The sudden change in the dynamics, in addition to other IR observables, indicates a structural transition. However, the results show that the cholesterol concentration at which the transition occurs is influenced by the curvature of the bilayers.
Enhanced water desalination performance through hierarchically-structured ceramic membranes

NARCIS (Netherlands)

Liu, Tong; Lei, Libin; Gu, Jianqiang; Wang, Yao; Winnubst, Louis; Chen, Chusheng; Ye, Chunsong; Chen, Fanglin

2017-01-01

Developments of membrane water desalination are impeded by low water vapor flux across the membrane. We present an innovative membrane design to significantly enhance the water vapor flux. A bilayer zirconia-based membrane with a thick hierarchically-structured support and a thin functional layer is
A portable lipid bilayer system for environmental sensing with a transmembrane protein.

Directory of Open Access Journals (Sweden)

Ryuji Kawano

Full Text Available This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin was achieved in the field at a high-altitude (∼3623 m. This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.
Effect of acetone accumulation on structure and dynamics of lipid membranes studied by molecular dynamics simulations.

Science.gov (United States)

Posokhov, Yevgen O; Kyrychenko, Alexander

2013-10-01

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

Energy Technology Data Exchange (ETDEWEB)

Wang Zunjing; Deserno, Markus, E-mail: zwang@cmu.ed, E-mail: deserno@andrew.cmu.ed [Department of Physics, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213 (United States)

2010-09-15

We study the lipid and phase transferability of our recently developed systematically coarse-grained solvent-free membrane model. The force field was explicitly parameterized to describe a fluid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer at 310 K with correct structure and area per lipid, while gaining at least three orders of magnitude in computational efficiency (see Wang and Deserno 2010 J. Phys. Chem. B 114 11207-20). Here, we show that exchanging CG tails, without any subsequent re-parameterization, creates reliable models of 1,2-dioleoylphosphatidylcholine (DOPC) and 1,2-dipalmitoylphosphatidylcholine (DPPC) lipids in terms of structure and area per lipid. Furthermore, all CG lipids undergo a liquid-gel transition upon cooling, with characteristics like those observed in experiments and all-atom simulations during phase transformation. These studies suggest a promising transferability of our force field parameters to different lipid species and thermodynamic state points, properties that are a prerequisite for even more complex systems, such as mixtures.
Systematic implicit solvent coarse-graining of bilayer membranes: lipid and phase transferability of the force field

International Nuclear Information System (INIS)

Wang Zunjing; Deserno, Markus

2010-01-01

We study the lipid and phase transferability of our recently developed systematically coarse-grained solvent-free membrane model. The force field was explicitly parameterized to describe a fluid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer at 310 K with correct structure and area per lipid, while gaining at least three orders of magnitude in computational efficiency (see Wang and Deserno 2010 J. Phys. Chem. B 114 11207-20). Here, we show that exchanging CG tails, without any subsequent re-parameterization, creates reliable models of 1,2-dioleoylphosphatidylcholine (DOPC) and 1,2-dipalmitoylphosphatidylcholine (DPPC) lipids in terms of structure and area per lipid. Furthermore, all CG lipids undergo a liquid-gel transition upon cooling, with characteristics like those observed in experiments and all-atom simulations during phase transformation. These studies suggest a promising transferability of our force field parameters to different lipid species and thermodynamic state points, properties that are a prerequisite for even more complex systems, such as mixtures.
Impact of amphiphilic molecules on the structure and stability of homogeneous sphingomyelin bilayer: Insights from atomistic simulations

Science.gov (United States)

Kumari, Pratibha; Kaur, Supreet; Sharma, Shobha; Kashyap, Hemant K.

2018-04-01

Modulation of lipid membrane properties due to the permeation of amphiphiles is an important biological process pertaining to many applications in the field of pharmaceutics, toxicology, and biotechnology. Sphingolipids are both structural and functional lipids that constitute an important component of mechanically stable and chemically resistant outer leaflets of plasma membranes. Here, we present an atomistic molecular dynamics simulation study to appreciate the concentration-dependent effects of small amphiphilic molecules, such as ethanol, acetone, and dimethyl sulfoxide (DMSO), on the structure and stability of a fully hydrated homogeneous N-palmitoyl-sphingomyelin (PSM) bilayer. The study reveals an increase in the lateral expansion of the bilayer along with disordering of the hydrophobic lipid tails on increasing the concentration of ethanol. At higher concentrations of ethanol, rupturing of the bilayer is quite evident through the analysis of partial electron density profiles and lipid tail order parameters. For ethanol containing systems, permeation of water molecules in the hydrophobic part of the bilayer is allowed through local defects made due to the entry of ethanol molecules via ethanol-ethanol and ethanol-PSM hydrogen bonds. Moreover, the extent of PSM-PSM hydrogen bonding decreases with increasing ethanol concentration. On the other hand, acetone and DMSO exhibit minimal effects on the stability of the PSM bilayer at their lower concentrations, but at higher concentrations they tend to enhance the stability of the bilayer. The simulated potential of mean force (PMF) profiles for the translocation of the three solutes studied reveal that the free-energy of transfer of an ethanol molecule across the PSM lipid head region is lower than that for acetone and DMSO molecules. However, highest free-energy rise in the core hydrophobic part of the bilayer is observed for the DMSO molecule, whereas the ethanol and acetone PMF profiles show a lower barrier in
Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

Energy Technology Data Exchange (ETDEWEB)

Straatsma, TP

2006-12-01

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

Efficient preparation and analysis of membrane and membrane protein systems

Czech Academy of Sciences Publication Activity Database

Javanainen, M.; Martinez-Seara, Hector

2016-01-01

Roč. 1858, č. 10 (2016), s. 2468-2482 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : tools and software * membrane building * protein insertion * molecular dynamics * lipid bilayer Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2016
Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

NARCIS (Netherlands)

Notman, Rebecca; Anwar, Jamshed; Briels, Willem J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO)
Molecular Origin of Model Membrane Bending Rigidity

International Nuclear Information System (INIS)

Kurtisovski, Erol; Taulier, Nicolas; Waks, Marcel; Ober, Raymond; Urbach, Wladimir

2007-01-01

The behavior of the bending modulus κ of bilayers in lamellar phases was studied by Small Angle X-ray Scattering technique for various nonionic C i E j surfactants. The bilayers are either unswollen and dispersed in water or swollen by water and dispersed in dodecane. For unswollen bilayers, the values of κ decrease with both an increase in the area per surfactant molecule and in the polar head length. They increase when the aliphatic chain length increases at constant area per surfactant molecule. Whereas for water-swollen membranes, the values of κ decrease as the content of water increases converging to the value of the single monolayer bending modulus. Such a behavior results from the decoupling of the fluctuations of the two surfactant membrane monolayers. Our results emphasize the determinant contribution of the surfactant conformation to κ
Solid-state nuclear magnetic resonance measurements of HIV fusion peptide 13CO to lipid 31P proximities support similar partially inserted membrane locations of the α helical and β sheet peptide structures.

Science.gov (United States)

Gabrys, Charles M; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D; Weliky, David P

2013-10-03

Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the ∼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a ∼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and β sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and
Mercury-Supported Biomimetic Membranes for the Investigation of Antimicrobial Peptides

Directory of Open Access Journals (Sweden)

Lucia Becucci

2014-01-01

Full Text Available Tethered bilayer lipid membranes (tBLMs consist of a lipid bilayer interposed between an aqueous solution and a hydrophilic “spacer” anchored to a gold or mercury electrode. There is great potential for application of these biomimetic membranes for the elucidation of structure-function relationships of membrane peptides and proteins. A drawback in the use of mercury-supported tBLMs with respect to gold-supported ones is represented by the difficulty in applying surface sensitive, spectroscopic and scanning probe microscopic techniques to gather information on the architecture of these biomimetic membranes. Nonetheless, mercury-supported tBLMs are definitely superior to gold-supported biomimetic membranes for the investigation of the function of membrane peptides and proteins, thanks to a fluidity and lipid lateral mobility comparable with those of bilayer lipid membranes interposed between two aqueous phases (BLMs, but with a much higher robustness and resistance to electric fields. The different features of mercury-supported tBLMs reconstituted with functionally active membrane proteins and peptides of bacteriological or pharmacological interest may be disclosed by a judicious choice of the most appropriate electrochemical techniques. We will describe the way in which electrochemical impedance spectroscopy, potential-step chronocoulometry, cyclic voltammetry and phase-sensitive AC voltammetry are conveniently employed to investigate the structure of mercury-supported tBLMs and the mode of interaction of antimicrobial peptides reconstituted into them.
On ripples and rafts: Curvature induced nanoscale structures in lipid membranes

International Nuclear Information System (INIS)

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

2014-01-01

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
Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers

KAUST Repository

Chu, Shidong

2010-11-01

A new approach for determining the membrane immersion depth of a spin-labeled probe has been developed using paramagnetic relaxation enhancement (PRE) in solid-state NMR spectroscopy. A DOXYL spin label was placed at different sites of 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC) phospholipid bilayers as paramagnetic moieties and the resulting enhancements of the longitudinal relaxation (T1) times of 31P nuclei on the surface of the bilayers were measured by a standard inversion recovery pulse sequence. The 31P NMR spin-lattice relaxation times decrease steadily as the DOXYL spin label moves closer to the surface as well as the concentration of the spin-labeled lipids increase. The enhanced relaxation vs. the position and concentration of spin-labels indicate that PRE induced by the DOXYL spin label are significant to determine longer distances over the whole range of the membrane depths. When these data were combined with estimated correlation times τc, the r-6-weighted, time-averaged distances between the spin-labels and the 31P nuclei on the membrane surface were estimated. The application of using this solid-state NMR PRE approach coupled with site-directed spin labeling (SDSL) may be a powerful method for measuring membrane protein immersion depth. © 2010 Elsevier Inc. All rights reserved.
A Molecular Dynamics Study of the Structural and Dynamical Properties of Putative Arsenic Substituted Lipid Bilayers

Directory of Open Access Journals (Sweden)

Ratna Juwita

2013-04-01

Full Text Available Cell membranes are composed mainly of phospholipids which are in turn, composed of five major chemical elements: carbon, hydrogen, nitrogen, oxygen, and phosphorus. Recent studies have suggested the possibility of sustaining life if the phosphorus is substituted by arsenic. Although this issue is still controversial, it is of interest to investigate the properties of arsenated-lipid bilayers to evaluate this possibility. In this study, we simulated arsenated-lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-arsenocholine (POAC, lipid bilayers using all-atom molecular dynamics to understand basic structural and dynamical properties, in particular, the differences from analogous 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, (POPC lipid bilayers. Our simulations showed that POAC lipid bilayers have distinct structural and dynamical properties from those of native POPC lipid bilayers. Relative to POPC lipid bilayers, POAC lipid bilayers have a more compact structure with smaller lateral areas and greater order. The compact structure of POAC lipid bilayers is due to the fact that more inter-lipid salt bridges are formed with arsenate-choline compared to the phosphate-choline of POPC lipid bilayers. These inter-lipid salt bridges bind POAC lipids together and also slow down the head group rotation and lateral diffusion of POAC lipids. Thus, it would be anticipated that POAC and POPC lipid bilayers would have different biological implications.
Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

Science.gov (United States)

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

2014-03-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.
Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone {sup 13}CO-{sup 15}N rotational-echo double-resonance solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Ujjayini; Xie Li; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

2013-02-15

The influenza virus fusion peptide is the N-terminal {approx}20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone {sup 13}CO-{sup 15}N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct {sup 13}C shifts.
/SIGMA PHI/-tocopherol: modifier of the phase state of the lipid bilayer

International Nuclear Information System (INIS)

Skrypin, V.I.; Bratkcovskaya, L.B.; Erin, A.N.; Kagan, V.E.

1985-01-01

This paper determines the action of low (near-physiological) concentrations of alpha-tocopherol on the character of the gel-liquid crystal transition in a lipid bilayer containing free fatty acids. Fifty mM of K + -phosphate buffer was made up in D 2 O and kept for several hours to enable substitution of H 1 by D 2, after which the buffer was dried and redissolved in D 2 O. The graphs of temperature dependence of relative signal intensity of protons of methylene groups in liposomes of different composition are presented. It is shown that the stabilizing action of alpha-tocopherol on the phase state of the lipid bilayer of membranes is one of the mechanisms by which the recently demonstrated ability of alpha-tocopherol to protect biological membranes against the injurious action of free fatty acids, through the formation of complexes of alpha-tocopherol with fatty acides, may be effected
Effect of Amphotericin B antibiotic on the properties of model lipid membrane

International Nuclear Information System (INIS)

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

2014-01-01

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
MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

Science.gov (United States)

Yamada, Yuma; Akita, Hidetaka; Kamiya, Hiroyuki; Kogure, Kentaro; Yamamoto, Takenori; Shinohara, Yasuo; Yamashita, Kikuji; Kobayashi, Hideo; Kikuchi, Hiroshi; Harashima, Hideyoshi

2008-02-01

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.
The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers

DEFF Research Database (Denmark)

Plasencia, Inés; Rivas, Luis; Keough, Kevin M W

2004-01-01

aggregation, and leakage of the aqueous content of the vesicles. The lipid-peptide interaction includes a significant hydrophobic component for both zwitterionic and anionic membranes, although the interaction with phosphatidylglycerol bilayers is also electrostatic in nature. The effects of the SP-C N......-terminal peptides on the membrane structure are mediated by significant perturbations of the packing order and mobility of phospholipid acyl chain segments deep in the bilayer, as detected by differential scanning calorimetry and spin-label ESR. These results suggest that the N-terminal region of SP-C, even...
Performance of a palladium membrane reactor using a Ni catalyst for fusion fuel impurities processing

International Nuclear Information System (INIS)

Willms, R.S.; Wilhelm, R.; Okuno, K.

1994-01-01

The palladium membrane reactor (PNM) provides a means to recover hydrogen isotopes from impurities expected to be present in fusion reactor exhaust. This recovery is based on reactions such as water-gas shift and steam reforming for which conversion is equilibrium limited. By including a selectively permeable membrane such as Pd/Ag in the catalyst bed, hydrogen isotopes can be removed from the reacting environment, thus promoting the reaction to complete conversion. Such a device has been built and operated at the Tritium Systems Test Assembly (TSTA) at Los Alamos National Laboratory (LANL). For the reactions listed above, earlier study with this unit has shown that hydrogen single-pass recoveries approaching 100% can be achieved. It was also determined that a nickel catalyst is a feasible choice for use with a PMR appropriate for fusion fuel impurities processing. The purpose of this study was to systematically assess the performance of the PMR using a nickel catalyst over a range of temperatures, feed compositions and flowrates. Reactions which were studied are the water-gas shift reaction and steam reforming
pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

Science.gov (United States)

Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Weber, Friedemann; Herold, Susanne; Pöhlmann, Stefan; Matrosovich, Mikhail

2017-06-01

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN
Neutron reflectivity study of substrate surface chemistry effects on supported phospholipid bilayer formation on (1120) sapphire.

Energy Technology Data Exchange (ETDEWEB)

Oleson, Timothy A. [University of Wisconsin, Madison; Sahai, Nita [University of Akron; Wesolowski, David J [ORNL; Dura, Joseph A [ORNL; Majkrzak, Charles F [ORNL; Giuffre, Anthony J. [University of Wisconsin, Madison

2012-01-01

Oxide-supported phospholipid bilayers (SPBs) used as biomimetric membranes are significant for a broad range of applications including improvement of biomedical devices and biosensors, and in understanding biomineralization processes and the possible role of mineral surfaces in the evolution of pre-biotic membranes. Continuous-coverage and/or stacjed SPBs retain properties (e.,g. fluidity) more similar to native biological membranes, which is desirable for most applications. Using neutron reflectivity, we examined face coverage and potential stacking of dipalmitoylphosphatidylcholine (DPPC) bilayers on the (1120) face of sapphire (a-Al2O3). Nearly full bilayers were formed at low to neutral pH, when the sapphire surface is positively charged, and at low ionic strength (l=15 mM NaCl). Coverage decreased at higher pH, close to the isoelectric point of sapphire, and also at high I>210mM, or with addition of 2mM Ca2+. The latter two effects are additive, suggesting that Ca2+ mitigates the effect of higher I. These trends agree with previous results for phospholipid adsorption on a-Al2O3 particles determined by adsorption isotherms and on single-crystal (1010) sapphire by atomic force microscopy, suggesting consistency of oxide surface chemistry-dependent effects across experimental techniques.
Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

Science.gov (United States)

Therrien, Alexandre; Fournier, Alain; Lafleur, Michel

2016-05-05

The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner
Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

NARCIS (Netherlands)

Ulrich, AS; Glabe, CG; Hoekstra, D

1998-01-01

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal
Nanoscale cell membrane organization : a near-field optical view

NARCIS (Netherlands)

Koopman, Marjolein

2006-01-01

The cell plasma membrane of eukaryotic cells is a lipid bi-layer that separates the cell cytosol from the extracellular environment. The composition and organization of proteins and lipids within this bi-layer have a direct impact on many cellular processes, since they form the senses of the cell.

pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

Directory of Open Access Journals (Sweden)

Priyanka Prakash

2015-10-01

Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.
Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

Directory of Open Access Journals (Sweden)

Bárbara Hissa

Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of
Interaction of Impulsive Pressures of Cavitation Bubbles with Cell Membranes during Sonoporation

Science.gov (United States)

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

2006-05-01

Ultrasound contrast agents (UCAs), are capable of enhancing non-invasive cytoplasmic molecular delivery in the presence of ultrasound. Collapse of UCAs may generate nano-scale cavitation bubbles, resulting in the transient permeabilization of the cell membrane. In the present study, we investigated the interaction of a cavitation bubble-induced shock wave with a cell membrane using acoustic theory and molecular dynamics (MD) simulation. From the theory, we obtained the shock wave propagation distance from the center of a cavitation bubble that would induce membrane damage. The MD simulation determined the relationship between the uptake of water molecules into the lipid bilayer and the shock wave. The interaction of the shock wave induced a structural change of the bilayer and subsequently increased the fluidity of each molecule. These changes in the bilayer due to shock waves may be an important factor in the use of UCAs to produce the transient membrane permeability during sonoporation.
Discriminating binding and positioning of amphiphiles to lipid bilayers by {sup 1}H NMR

Energy Technology Data Exchange (ETDEWEB)

Evanics, F. [Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North Mississauga, Ont., L5L 1C6 (Canada); Prosser, R.S. [Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North Mississauga, Ont., L5L 1C6 (Canada)]. E-mail: sprosser@utm.utoronto.ca

2005-04-04

The binding and positioning in lipid bilayers of three well-known drugs--imipramine, nicotine, and caffeine--have been studied using {sup 1}H NMR. The membrane model system consisted of 'fast-tumbling' lipid bicelles, in which a bilayered lipid domain, composed of the unsaturated lipid, 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine (DMLPC) was surrounded by a rim of deuterated detergent-like lipids, consisting of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC-d22). Binding and immersion depth information was obtained by three experiments. (1) {sup 1}H chemical shift perturbations, upon transfer of the amphiphiles from water to a bicelle mixture, were used to estimate regions of the amphiphiles that interact with the membrane. (2) Water contact to resolvable protons was measured through a Nuclear Overhauser Effect (NOE) between water and resolvable drug and lipid resonances. In the case of both lipids and membrane bound drugs, positive NOEs with large cross-relaxation rates were measured for most resonances originating from the membrane hydrophilic region, while negative NOEs were observed predominantly to resonances in the hydrophobic region of the membrane. (3) {sup 1}H NMR measurements of oxygen-induced (paramagnetic) spin-lattice relaxation rates, which are known to increase with membrane immersion depth, were used to corroborate conclusions based on chemical shift perturbations and water-ligand NOEs.
A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

International Nuclear Information System (INIS)

Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

2010-01-01

We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.
Proton and carbon-13 nuclear magnetic resonance studies of the effects of retinal on the dynamic structure and stability of lipid bilayer

International Nuclear Information System (INIS)

Inoue, Yoshio; Hanafusa, Yoshito; Toda, Masakazu; Chujo, Riichiro

1982-01-01

The effects of retinal and vitamin A on the dynamic structure and stability of hen egg yolk lecithin bilayers have been studied by means of carbon-13 and proton NMR spectroscopies. 13 C spin-lattice relaxation and paramagnetic ion permeability studies on lecithin bilayers indicate a marked decrease in flexibility of the lipid acyl chain and a breakdown of membrane impermeableness to ion by the intercalated all-trans- and 11-cis-retinal, whereas the effect of incorporated vitamin A on the fluidity of bilayers is small and its impermeableness to ion remains effective even in the presence of higher concentration of vitamin A. The experimental results are discussed in connection with the mechanism of the permeability change in photoreceptive disk membrane. (author)
Osteoclast Fusion

DEFF Research Database (Denmark)

Marie Julie Møller, Anaïs; Delaissé, Jean-Marie; Søe, Kent

2017-01-01

on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono-nucleated pre-osteoclasts, syncytin-1 promotes fusion of two multi-nucleated osteoclasts, but also reduces the number of fusions between mono-nucleated pre-osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through...... individual fusion events using time-lapse and antagonists of CD47 and syncytin-1. All time-lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin-1 have different roles in osteoclast fusion depending...... broad contact surfaces between the partners' cell membrane while syncytin-1 mediate fusion through phagocytic-cup like structure. J. Cell. Physiol. 9999: 1-8, 2016. © 2016 Wiley Periodicals, Inc....
Molecular dynamics simulations of the effects of sodium dodecyl sulfate on lipid bilayer

International Nuclear Information System (INIS)

Xu Bin; Lin Wen-Qiang; Wang Xiao-Gang; Zhou Guo-Quan; Chen Jun-Lang; Zeng Song-wei

2017-01-01

Molecular dynamics simulations have been performed on the fully hydrated lipid bilayer with different concentrations of sodium dodecyl sulfate (SDS). SDS can readily penetrate into the membrane. The insertion of SDS causes a decrease in the bilayer area and increases in the bilayer thickness and lipid tail order, when the fraction of SDS is less than 28%. Through calculating the binding energy, we confirm that the presence of SDS strengthens the interactions among the DPPC lipids, while SDS molecules act as intermedia. Both the strong hydrophilic interactions between sulfate and phosphocholine groups and the hydrophobic interactions between SDS and DPPC hydrocarbon chains contribute to the tight packing and ordered alignment of the lipids. These results are in good agreement with the experimental observations and provide atomic level information that complements the experiments. (paper)
A criterion to identify the equilibration time in lipid bilayer simulations

Directory of Open Access Journals (Sweden)

Rodolfo D. Porasso

2012-11-01

Full Text Available With the aim of establishing a criterion for identifying when a lipid bilayer has reached steady state using the molecular dynamics simulation technique, lipid bilayers of different composition in their liquid crystalline phase were simulated in aqueous solution in presence of CaCl_2 as electrolyte, at different concentration levels. In this regard, we used two different lipid bilayer systems: one composed by 288 DPPC (DiPalmitoylPhosphatidylCholine and another constituted by 288 DPPS (DiPalmitoylPhosphatidylSerine. In this sense, for both type of lipid bilayers, we have studied the temporal evolution of some lipids properties, such as the surface area per lipid, the deuterium order parameter, the lipid hydration and the lipid-calcium coordination. From their analysis, it became evident how each property has a different time to achieve equilibrium. The following order was found, from faster property to slower property: coordination of ions $approx$ deuterium order parameter > area per lipid $approx$ hydration. Consequently, when the hydration of lipids or the mean area per lipid are stable, we can ensure that the lipid membrane has reached the steady state.
Protein-lipid interactions: from membrane domains to cellular networks

National Research Council Canada - National Science Library

Tamm, Lukas K

2005-01-01

... membranes is the lipid bilayer. Embedded in the fluid lipid bilayer are proteins of various shapes and traits. This volume illuminates from physical, chemical and biological angles the numerous - mostly quite weak - interactions between lipids, proteins, and proteins and lipids that define the delicate, highly dynamic and yet so stable fabri...
Stability of asymmetric lipid bilayers assessed by molecular dynamics simulations

NARCIS (Netherlands)

Esteban-Martin, Santi; Risselada, H. Jelger; Salgado, Jesus; Marrink, Siewert J.

2009-01-01

The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted
Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

Science.gov (United States)

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

2015-10-07

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

NARCIS (Netherlands)

van Weerd, Jasper; Krabbenborg, Sven; Eijkel, Jan C.T.; Karperien, Hermanus Bernardus Johannes; Huskens, Jurriaan; Jonkheijm, Pascal

2014-01-01

A micro supported lipid bilayer (SLB) electrophoresis method was developed, which functions at low potentials and appreciable operating times. To this end, (hydroxymethyl)-ferrocene (FcCH2OH) was employed to provide an electrochemical reaction at the anode and cathode at low applied potential to
Probing lipid membrane electrostatics

Science.gov (United States)

Yang, Yi

The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive manner will be useful in identifying the biological effects of the dipole potential. Finally, heterogeneous model membranes were studied with fluid electric force microscopy (FEFM). Electrostatic mapping was demonstrated with 50 nm resolution. The capabilities of quantitative electrostatic measurement and lateral charge density mapping make AFM a unique and powerful
How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

Science.gov (United States)

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

2012-02-01

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

Science.gov (United States)

Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

2013-05-01

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Copyright © 2013 Wiley Periodicals, Inc.
Drug membrane interaction and the importance for drug transport, distribution, accumulation, efficacy and resistance.

Science.gov (United States)

Seydel, J K; Coats, E A; Cordes, H P; Wiese, M

1994-10-01

Some aspects of drug membrane interaction and its influence on drug transport, accumulation, efficacy and resistance have been discussed. The interactions manifest themselves macroscopically in changes in the physical and thermodynamic properties of "pure membranes" or bilayers. As various amounts of foreign molecules enter the membrane, in particular the main gel to liquid crystalline phase transition can be dramatically changed. This may change permeability, cell-fusion, cell resistance and may also lead to changes in conformation of the embedded receptor proteins. Furthermore, specific interactions with lipids may lead to drug accumulation in membranes and thus to much larger concentrations at the active site than present in the surrounding water phase. The lipid environment may also lead to changes in the preferred conformation of drug molecules. These events are directly related to drug efficacy. The determination of essential molecular criteria for the interaction could be used to design new and more selective therapeutics. This excursion in some aspects of drug membrane interaction underlines the importance of lipids and their interaction with drug molecules for our understanding of drug action, but this is not really a new thought but has been formulated in 1884 by THUDICUM: "Phospholipids are the centre, life and chemical soul of all bioplasm whatsoever, that of plants as well as of animals".
Protein-induced bilayer Perturbations: Lipid ordering and hydrophobic coupling

DEFF Research Database (Denmark)

Petersen, Frederic Nicolas Rønne; Laursen, Ib; Bohr, Henrik

2009-01-01

The host lipid bilayer is increasingly being recognized as an important non-specific regulator of membrane protein function. Despite considerable progress the interplay between hydrophobic coupling and lipid ordering is still elusive. We use electron spin resonance (ESR) to study the interaction...... between the model protein gramicidin and lipid bilayers of varying thickness. The free energy of the interaction is up to −6 kJ/mol; thus not strongly favored over lipid–lipid interactions. Incorporation of gramicidin results in increased order parameters with increased protein concentration...... and hydrophobic mismatch. Our findings also show that at high protein:lipid ratios the lipids are motionally restricted but not completely immobilized. Both exchange on and off rate values for the lipid ↔ gramicidin interaction are lowest at optimal hydrophobic matching. Hydrophobic mismatch of few Å results...
Composition, structure and properties of POPC–triolein mixtures. Evidence of triglyceride domains in phospholipid bilayers

DEFF Research Database (Denmark)

Duelund, Lars; Jensen, Grethe Vestergaard; Hannibal-Bach, Hans Kristian

2013-01-01

We have in this study investigated the composition, structure and spectroscopical properties of multilamellar vesicles composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and up to 10mol% of triolein (TO), a triglyceride. We found in agreement with previous results...... as vesicular structures containing entrapped water. Bilayer structure of the membranes was supported by small angle X-ray scattering that showed the membranes to form a lamellar phase. Fluorescence spectroscopy with the polarity sensitive dye Nile red revealed, that the LF samples with more than 5mol......% TO contained pure TO domains. These observations are consistent with an earlier MD simulation study by us and our co-workers suggesting triglycerides to be located in lens shaped, blister-like domains between the two lipid bilayer leaflets (Khandelia et al. (2010) [26])....
Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

Science.gov (United States)

Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

2011-03-01

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

International Nuclear Information System (INIS)

Martín-García, Fernando; Mendieta-Moreno, Jesús Ignacio; Mendieta, Jesús; Gómez-Puertas, Paulino

2012-01-01

Highlights: ► Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. ► HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. ► HRS domains of F protein form three single α-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from β-sheet conformation to an elongated coil and then spontaneously to an α-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.
Measuring the composition-curvature coupling in binary lipid membranes by computer simulations

International Nuclear Information System (INIS)

Barragán Vidal, I. A.; Müller, M.; Rosetti, C. M.; Pastorino, C.

2014-01-01

The coupling between local composition fluctuations in binary lipid membranes and curvature affects the lateral membrane structure. We propose an efficient method to compute the composition-curvature coupling in molecular simulations and apply it to two coarse-grained membrane models—a minimal, implicit-solvent model and the MARTINI model. Both the weak-curvature behavior that is typical for thermal fluctuations of planar bilayer membranes as well as the strong-curvature regime corresponding to narrow cylindrical membrane tubes are studied by molecular dynamics simulation. The simulation results are analyzed by using a phenomenological model of the thermodynamics of curved, mixed bilayer membranes that accounts for the change of the monolayer area upon bending. Additionally the role of thermodynamic characteristics such as the incompatibility between the two lipid species and asymmetry of composition are investigated
Measuring the composition-curvature coupling in binary lipid membranes by computer simulations

Energy Technology Data Exchange (ETDEWEB)

Barragán Vidal, I. A., E-mail: vidal@theorie.physik.uni-goettingen.de; Müller, M., E-mail: mmueller@theorie.physik.uni-goettingen.de [Institut für Theoretische Physik, Georg-August-Universität, Friedrich-Hund-Platz 1, 37077 Göttingen (Germany); Rosetti, C. M., E-mail: carla@dqb.fcq.unc.edu.ar [Centro de Investigaciones en Química Biológica de Córdoba, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba (Argentina); Pastorino, C., E-mail: pastor@cnea.gov.ar [Departamento de Física de la Materia Condensada, Centro Atómico Constituyentes, CNEA/CONICET, Av. Gral. Paz 1499, 1650 Pcia. de Buenos Aires (Argentina)

2014-11-21

The coupling between local composition fluctuations in binary lipid membranes and curvature affects the lateral membrane structure. We propose an efficient method to compute the composition-curvature coupling in molecular simulations and apply it to two coarse-grained membrane models—a minimal, implicit-solvent model and the MARTINI model. Both the weak-curvature behavior that is typical for thermal fluctuations of planar bilayer membranes as well as the strong-curvature regime corresponding to narrow cylindrical membrane tubes are studied by molecular dynamics simulation. The simulation results are analyzed by using a phenomenological model of the thermodynamics of curved, mixed bilayer membranes that accounts for the change of the monolayer area upon bending. Additionally the role of thermodynamic characteristics such as the incompatibility between the two lipid species and asymmetry of composition are investigated.
Permeability of a Fluid Lipid Bilayer to Short-Chain Alcohols from First Principles.

Science.gov (United States)

Comer, Jeffrey; Schulten, Klaus; Chipot, Christophe

2017-06-13

Computational prediction of membrane permeability to small molecules requires accurate description of both the thermodynamics and kinetics underlying translocation across the lipid bilayer. In this contribution, well-converged, microsecond-long free-energy calculations are combined with a recently developed subdiffusive kinetics framework to describe the membrane permeation of a homologous series of short-tail alcohols, from methanol to 1-butanol, with unprecedented fidelity to the underlying molecular models. While the free-energy profiles exhibit barriers for passage through the center of the bilayer in all cases, the height of these barriers decreases with the length of the aliphatic chain of the alcohol, in quantitative agreement with experimentally determined differential solvation free energies in water and oil. A unique aspect of the subdiffusive model employed herein, which was developed in a previous article, is the determination of a position-dependent fractional order which quantifies the degree to which the motion of the alcohol deviates from classical diffusion along the thickness of the membrane. In the aqueous medium far from the bilayer, this quantity approaches 1.0, the asymptotic limit for purely classical diffusion, whereas it dips below 0.75 near the center of the membrane irrespective of the permeant. Remarkably, the fractional diffusivity near the center of membrane, where its influence on the permeability is the greatest, is similar among the four permeants despite the large difference in molecular weight and lipophilicity between methanol and 1-butanol. The relative permeabilities, which are estimated from the free-energy and fractional diffusivity profiles, are therefore determined predominantly by differences in the former rather than the latter. The predicted relative permeabilities are highly correlated with existing experimental results, albeit they do not agree quantitatively with them. On the other hand, quite unexpectedly, the
Dynamic behavior of ultra large graphene-based membranes using electrothermal transduction

Science.gov (United States)

Al-mashaal, A. K.; Wood, G. S.; Torin, A.; Mastropaolo, E.; Newton, M. J.; Cheung, R.

2017-12-01

This letter reports an experimental study of an electrothermal actuator made from an ultra-large graphene-based bilayer thin film with a diameter to thickness aspect ratio of ˜10 000. Suspended thin films consisting of multilayer graphene and 350-500 nm-thick Poly(methyl methacrylate) have been transferred over circular cavities with a diameter of 3.5 mm. The use of bilayer materials with different mechanical and thermal properties results in thin film structures that can be induced to vibrate mechanically under the electrothermal transduction mechanism. The dynamic response of the bilayer has been investigated electrothermally by driving the structures with a combination of alternating current and direct current actuation voltages ( Va c and Vd c) and characterizing their resonant frequencies. It has been found that the bilayer thin film structure behaves as a membrane. In addition, the actuation configurations affect not only the amplitude of vibration but also the tuning of the resonant frequency of the vibrating membranes. The existence of Joule heating-induced tension lowers the mechanical stiffness of the membrane and hence shifts the resonant frequency downwards by -108187 ppm. A resonant frequency of 3.26 kHz with a vibration amplitude of 4.34 nm has been achieved for 350 nm-thick membranes under actuation voltages of 1 V of Va c and 8 V of Vd c.
Self-assembly of tissue spheroids on polymeric membranes.

Science.gov (United States)

Messina, Antonietta; Morelli, Sabrina; Forgacs, Gabor; Barbieri, Giuseppe; Drioli, Enrico; De Bartolo, Loredana

2017-07-01

In this study, multicellular tissue spheroids were fabricated on polymeric membranes in order to accelerate the fusion process and tissue formation. To this purpose, tissue spheroids composed of three different cell types, myoblasts, fibroblasts and neural cells, were formed and cultured on agarose and membranes of polycaprolactone (PCL) and chitosan (CHT). Membranes prepared by a phase-inversion technique display different physicochemical, mechanical and transport properties, which can affect the fusion process. The membranes accelerated the fusion process of a pair of spheroids with respect to the inert substrate. In this process, a critical role is played by the membrane properties, especially by their mechanical characteristics and oxygen and carbon dioxide mass transfer. The rate of fusion was quantified and found to be similar for fibroblast, myoblast and neural tissue spheroids on membranes, which completed the fusion within 3 days. These spheroids underwent faster fusion and maturation on PCL membrane than on agarose, the rate of fusion being proportional to the value of oxygen and carbon dioxide permeances and elastic characteristics. Consequently, tissue spheroids on the membranes expressed high biological activity in terms of oxygen uptake, making them more suitable as building blocks in the fabrication of tissues and organs. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
Protection of DPPC phospholipid liposomal membrane against radiation oxidative damage by antioxidants

Energy Technology Data Exchange (ETDEWEB)

Marathe, D.L.; Pandey, B.N.; Mishra, K.P [Bhabha Atomic Research Centre, Mumbai (India)

2000-05-01

Investigations in our laboratory on egg lecithin liposomes have recently showed a marked protection against damage by gamma radiation when cholesterol was present in the composition of vesicles suggesting a role of bilayer molecular architecture in the mechanism of free radical mediated lipid peroxidation. Present study was designed to determine the changes in bilayer permeability in DPPC unilamelar vesicles after exposure to gamma radiation by monitoring the leakage of pre-loaded carboxyfluorescein (CF), a marker loaded in aqueous interior of vesicle and fluidity alterations in the bilayer using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a membrane bilayer probe. It was found that radiation doses of an order of magnitude higher were required to produce detectable changes in vesicles of DPPC than in the vesicles of egg lecithin suggesting a modulating role of chemical nature of composition in the membrane radiation sensitivity. It was significant to find that the leakage of CF from and incorporation of DPH into vesicle bilayer showed similar response pattern to radiation doses (0.1-6 kGy) which was also found to be dose rate dependent. Presence of antioxidants; alpha-tocopherol (0.15 mole %) in the bilayer membrane or ascorbic acid (0.1 mM) in the aqueous region significantly protected DPPC vesicles from radiation damage as determined from DPH uptake kinetics suggesting involvement of reactive free radicals of lipids as well as water radicals in the mechanism of membrane peroxidative damage. The magnitude of protection was found to increase with the increasing concentration of both these antioxidants but comparisons showed that {alpha}-tocopherol was far more effective in protecting the vesicles than ascorbic acid. These results contribute to our understanding of the mechanism of radiation oxidative damage and its modification by radical scavenging and/or organizational modulation which emphasize the importance of structure and composition of
Protection of DPPC phospholipid liposomal membrane against radiation oxidative damage by antioxidants

International Nuclear Information System (INIS)

Marathe, D.L.; Pandey, B.N.; Mishra, K.P

2000-01-01

Investigations in our laboratory on egg lecithin liposomes have recently showed a marked protection against damage by gamma radiation when cholesterol was present in the composition of vesicles suggesting a role of bilayer molecular architecture in the mechanism of free radical mediated lipid peroxidation. Present study was designed to determine the changes in bilayer permeability in DPPC unilamelar vesicles after exposure to gamma radiation by monitoring the leakage of pre-loaded carboxyfluorescein (CF), a marker loaded in aqueous interior of vesicle and fluidity alterations in the bilayer using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a membrane bilayer probe. It was found that radiation doses of an order of magnitude higher were required to produce detectable changes in vesicles of DPPC than in the vesicles of egg lecithin suggesting a modulating role of chemical nature of composition in the membrane radiation sensitivity. It was significant to find that the leakage of CF from and incorporation of DPH into vesicle bilayer showed similar response pattern to radiation doses (0.1-6 kGy) which was also found to be dose rate dependent. Presence of antioxidants; alpha-tocopherol (0.15 mole %) in the bilayer membrane or ascorbic acid (0.1 mM) in the aqueous region significantly protected DPPC vesicles from radiation damage as determined from DPH uptake kinetics suggesting involvement of reactive free radicals of lipids as well as water radicals in the mechanism of membrane peroxidative damage. The magnitude of protection was found to increase with the increasing concentration of both these antioxidants but comparisons showed that α-tocopherol was far more effective in protecting the vesicles than ascorbic acid. These results contribute to our understanding of the mechanism of radiation oxidative damage and its modification by radical scavenging and/or organizational modulation which emphasize the importance of structure and composition of
Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

Directory of Open Access Journals (Sweden)

Katie D. Hickey

2011-01-01

Full Text Available Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+ K+-ATPase or -amylase. Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+ K+-ATPase within the membranes.
Localization and Orientation of Xanthophylls in a Lipid Bilayer.

Science.gov (United States)

Grudzinski, Wojciech; Nierzwicki, Lukasz; Welc, Renata; Reszczynska, Emilia; Luchowski, Rafal; Czub, Jacek; Gruszecki, Wieslaw I

2017-08-29

Xanthophylls (polar carotenoids) play diverse biological roles, among which are modulation of the physical properties of lipid membranes and protection of biomembranes against oxidative damage. Molecular mechanisms underlying these functions are intimately related to the localization and orientation of xanthophyll molecules in lipid membranes. In the present work, we address the problem of localization and orientation of two xanthophylls present in the photosynthetic apparatus of plants and in the retina of the human eye, zeaxanthin and lutein, in a single lipid bilayer membrane formed with dimyristoylphosphatidylcholine. By using fluorescence microscopic analysis and Raman imaging of giant unilamellar vesicles, as well as molecular dynamics simulations, we show that lutein and zeaxanthin adopt a very similar transmembrane orientation within a lipid membrane. In experimental and computational approach, the average tilt angle of xanthophylls relative to the membrane normal is independently found to be ~40 deg, and results from hydrophobic mismatch between the membrane thickness and the distance between the terminal hydroxyl groups of the xanthophylls. Consequences of such a localization and orientation for biological activity of xanthophylls are discussed.
cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

Science.gov (United States)

Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.
Transmembrane peptides as sensors of the membrane physical state

Science.gov (United States)

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

2018-05-01

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

Energy Technology Data Exchange (ETDEWEB)

Lenné, T.; Kent, B.; Koster, K.L.; Garvey, C.J.; Bryant, G. (ANSTO); (USD); (ANU); (RMIT)

2012-02-06

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

Science.gov (United States)

Wyatt, Debra L; de Godoy, Carlos Marcelo G; Cukierman, Samuel

2009-05-14

The transfer of protons (H+) in gramicidin (gA) channels is markedly distinct in monoglyceride and phospholipid membranes. In this study, the molecular groups that account for those differences were investigated using a new methodology. The rates of H+ transfer were measured in single gA channels reconstituted in membranes made of plain ceramides or sphingomyelins and compared to those in monoglyceride and phospholipid bilayers. Single-channel conductances to protons (gH) were significantly larger in sphingomyelin than in ceramide membranes. A novel and unsuspected finding was that H+ transfer was heavily attenuated or completely blocked in ceramide (but not in sphingomyelin) membranes in low-ionic-strength solutions. It is reasoned that H-bond dynamics at low ionic strengths between membrane ceramides and gA makes channels dysfunctional. The rate of H+ transfer in gA channels in ceramide membranes is significantly higher than that in monoglyceride bilayers. This suggests that solvation of the hydrophobic surface of gA channels by two acyl chains in ceramides stabilizes the gA channels and the water wire inside the pore, leading to an enhancement of H+ transfer in relation to that occurring in monoglyceride membranes. gH values in gA channels are similar in ceramide and monoglyceride bilayers and in sphingomyelin and phospholipid membranes. It is concluded that phospho headgroups in membranes have significant effects on the rate of H+ transfer at the membrane gA channel/solution interfaces, enhancing the entry and exit rates of protons in channels.
NIR studies of cholesterol-dependent structural modification of the model lipid bilayer doped with inhalation anesthetics

Science.gov (United States)

Kuć, Marta; Cieślik-Boczula, Katarzyna; Rospenk, Maria

2018-06-01

The influence of cholesterol on the structure of the model lipid bilayers treated with inhalation anesthetics (enflurane, isoflurane, sevoflurane and halothane) was investigated employing near-infrared (NIR) spectroscopy combined with the Principal Component Analysis (PCA). The conformational changes occurring in the hydrophobic area of the lipid bilayers were analyzed using the first overtones of symmetric (2νs) and antisymmetric (2νas) stretching vibrations of the CH2 groups of lipid aliphatic chains. The temperature values of chain-melting phase transition (Tm) of anesthetic-mixed dipalmitoylphosphatidylcholine (DPPC)/cholesterol and dipalmitoylphosphatidylglycerol (DPPG)/cholesterol membranes, which were obtained from the PCA analysis, were compared with cholesterol-free DPPC and DPPG bilayers mixed with inhalation anesthetics.
Nano-swimmers in biological membranes and propulsion hydrodynamics in two dimensions.

Science.gov (United States)

Huang, Mu-Jie; Chen, Hsuan-Yi; Mikhailov, Alexander S

2012-11-01

Active protein inclusions in biological membranes can represent nano-swimmers and propel themselves in lipid bilayers. A simple model of an active inclusion with three particles (domains) connected by variable elastic links is considered. First, the membrane is modeled as a two-dimensional viscous fluid and propulsion behavior in two dimensions is examined. After that, an example of a microscopic dynamical simulation is presented, where the lipid bilayer structure of the membrane is resolved and the solvent effects are included by multiparticle collision dynamics. Statistical analysis of data reveals ballistic motion of the swimmer, in contrast to the classical diffusion behavior found in the absence of active transitions between the states.
Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

Czech Academy of Sciences Publication Activity Database

Javanainen, M.; Martinez-Seara, Hector; Metzler, R.; Vattulainen, I.

2017-01-01

Roč. 8, č. 17 (2017), s. 4308-4313 ISSN 1948-7185 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : giant unilamellar vesicles * single-molecule tracking * lipid bilayer membranes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 9.353, year: 2016
Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

Science.gov (United States)

Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

2007-06-12

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.
Mixed bilayer containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine: lipid complexation, ion binding, and electrostatics.

Science.gov (United States)

Pandit, Sagar A; Bostick, David; Berkowitz, Max L

2003-11-01

Two mixed bilayers containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine at a ratio of 5:1 are simulated in NaCl electrolyte solutions of different concentration using the molecular dynamics technique. Direct NH.O and CH.O hydrogen bonding between lipids was observed to serve as the basis of interlipid complexation. It is deduced from our results and previous studies that dipalmitoylphosphatidylcholine alone is less likely to form interlipid complexes than in the presence of bound ions or other bilayer "impurities" such as dipalmitoylphosphatidylserine. The binding of counterions is observed and quantitated. Based upon the calculated ion binding constants, the Gouy-Chapman surface potential (theta) is calculated. In addition we calculated the electrostatic potential profile (Phi) by twice integrating the system charge distribution. A large discrepancy between and the value of Phi at the membrane surface is observed. However, at "larger" distance from the bilayer surface, a qualitative similarity in the z-profiles of Phi and psi(GC) is seen. The discrepancy between the two potential profiles near the bilayer surface is attributed to the discrete and nonbulk-like nature of water in the interfacial region and to the complex geometry of this region.
Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery.

Science.gov (United States)

Farzan, Shohreh F; Palermo, Laura M; Yokoyama, Christine C; Orefice, Gianmarco; Fornabaio, Micaela; Sarkar, Aurijit; Kellogg, Glen E; Greengard, Olga; Porotto, Matteo; Moscona, Anne

2011-11-04

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.

Model Biological Membranes and Possibilities of Application of Electrochemical Impedance Spectroscopy for Their Characterization

Czech Academy of Sciences Publication Activity Database

Skalová, Štěpánka; Vyskočil, V.; Barek, J.; Navrátil, Tomáš

2018-01-01

Roč. 30, č. 2 (2018), s. 207-219 ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA17-05387S; GA ČR GA17-03868S Institutional support: RVO:61388955 Keywords : Electrochemical impedance spectroscopy (EIS) * Liposomes * Model membrane * Phospholipid bilayer * Planar lipid bilayer * Supported lipid bilayers * Tethered lipid bilayers Subject RIV: CG - Electrochemistry OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 2.851, year: 2016
Interactions of Borneol with DPPC Phospholipid Membranes: A Molecular Dynamics Simulation Study

Directory of Open Access Journals (Sweden)

Qianqian Yin

2014-11-01

Full Text Available Borneol, known as a “guide” drug in traditional Chinese medicine, is widely used as a natural penetration enhancer in modern clinical applications. Despite a large number of experimental studies on borneol’s penetration enhancing effect, the molecular basis of its action on bio-membranes is still unclear. We carried out a series of coarse-grained molecular dynamics simulations with the borneol concentration ranging from 3.31% to 54.59% (v/v, lipid-free basis to study the interactions of borneol with aDPPC(1,2-dipalmitoylsn-glycero-3-phosphatidylcholine bilayer membrane, and the temperature effects were also considered. At concentrations below 21.89%, borneol’s presence only caused DPPC bilayer thinning and an increase in fluidity; A rise in temperature could promote the diffusing progress of borneol. When the concentration was 21.89% or above, inverted micelle-like structures were formed within the bilayer interior, which led to increased bilayer thickness, and an optimum temperature was found for the interaction of borneol with the DPPC bilayer membrane. These findings revealed that the choice of optimal concentration and temperature is critical for a given application in which borneol is used as a penetration enhancer. Our results not only clarify some molecular basis for borneol’s penetration enhancing effects, but also provide some guidance for the development and applications of new preparations containing borneol.
Salt-induced effects on natural and inverse DPPC lipid membranes: Molecular dynamics simulation.

Science.gov (United States)

Rezaei Sani, Seyed Mojtaba; Akhavan, Mojdeh; Jalili, Seifollah

2018-08-01

Molecular dynamics (MD) simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer and its neutral inverse-phosphocholine equivalent (DPCPe) were performed to find salt-induced effects on their surface structure and the nature of ion-lipid interactions. We found that the area per lipid is not considerably affected by the inversion, but the deuterium order parameter of carbon atoms in the region of carbonyl carbons changes dramatically. MD simulations indicate that Ca 2+ ions can bind to the surface of both DPPC and DPCPe membranes, but K + ions do not bind to them. In the case of Na + , however, the ions can bind to natural lipids but not to the inverse ones. Also, our results demonstrate that the hydration level of CPe bilayers is substantially lower than PC bilayers and the averaged orientation of water dipoles in the region of CPe headgroups is effectively inverted compared to PC lipids. This might be important in the interaction of the bilayer with its biological environment. Furthermore, it was found for the CPe bilayers that the enhanced peaks of the electrostatic potential profiles shift further away from the bilayer center relative to those of PC bilayers. This behavior makes the penetration of cations into the bilayer more difficult and possibly explains the experimentally observed enhanced release rates of anionic compounds in the CPe membrane. Copyright © 2018 Elsevier B.V. All rights reserved.
Fusomorphogenesis: cell fusion in organ formation.

Science.gov (United States)

Shemer, G; Podbilewicz, B

2000-05-01

Cell fusion is a universal process that occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. Very little is known about the molecular and cellular mechanisms of cell fusion during pattern formation. Here we review the dynamic anatomy of all cell fusions during embryonic and postembryonic development in an organism. Nearly all the cell fates and cell lineages are invariant in the nematode C. elegans and one third of the cells that are born fuse to form 44 syncytia in a reproducible and stereotyped way. To explain the function of cell fusion in organ formation we propose the fusomorphogenetic model as a simple cellular mechanism to efficiently redistribute membranes using a combination of cell fusion and polarized membrane recycling during morphogenesis. Thus, regulated intercellular and intracellular membrane fusion processes may drive elongation of the embryo as well as postembryonic organ formation in C. elegans. Finally, we use the fusomorphogenetic hypothesis to explain the role of cell fusion in the formation of organs like muscles, bones, and placenta in mammals and other species and to speculate on how the intracellular machinery that drive fusomorphogenesis may have evolved.
Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties

Science.gov (United States)

Montal, M.; Mueller, P.

1972-01-01

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

Science.gov (United States)

Carlo Reis, Emily C; Borges, Andréa P B; Araújo, Michel V F; Mendes, Vanessa C; Guan, Limin; Davies, John E

2011-12-01

The regeneration of tissues affected by periodontal disease is a complex process; it encompasses the formation of bone, cementum and periodontal ligament. We developed a semi-rigid PLGA (polylactide-co-glycolide acid)/CaP (calcium phosphate) bilayered biomaterial construct to promote periodontal regeneration, which has a continuous outer barrier membrane and an inner topographically complex component. Our experimental model compared periodontal prophylaxis alone with prophylaxis and biomaterial implantation in the treatment of class II furcation defects in dogs. Clinical evaluation, micro-computed tomography, histology and backscattered electron imaging were used for data analysis. Healing occurred uneventfully and bone volumetric values, trabecular number and trabecular thickness were all significantly greater in the treated group; while trabecular separation was significantly greater in the control group. New cementum, bone, and periodontal ligament with Sharpey fibre insertions were only seen in the treated group. Although periodontal regeneration has been reported elsewhere, the advantages of employing our bilayered PLGA + CaP construct are twofold: 1)it did not collapse into the defect; and, 2) its inner side was able to retain the blood clot throughout the buccal defect. The result was greater periodontal regeneration than has previously been reported with traditional flexible membranes. Copyright © 2011 Elsevier Ltd. All rights reserved.
Single-component supported lipid bilayers probed using broadband nonlinear optics.

Science.gov (United States)

Olenick, Laura L; Chase, Hilary M; Fu, Li; Zhang, Yun; McGeachy, Alicia C; Dogangun, Merve; Walter, Stephanie R; Wang, Hong-Fei; Geiger, Franz M

2018-01-31

Broadband SFG spectroscopy is shown to offer considerable advantages over scanning systems in terms of signal-to-noise ratios when probing well-formed single-component supported lipid bilayers formed from zwitterionic lipids with PC headgroups. The SFG spectra obtained from bilayers formed from DOPC, POPC, DLPC, DMPC, DPPC and DSPC show a common peak at ∼2980 cm -1 , which is subject to interference between the C-H and the O-H stretches from the aqueous phase, while membranes having transition temperatures above the laboratory temperature produce SFG spectra with at least two additional peaks, one at ∼2920 cm -1 and another at ∼2880 cm -1 . The results validate spectroscopic and structural data from SFG experiments utilizing asymmetric bilayers in which one leaflet differs from the other in the extent of deuteration. Differences in H 2 O-D 2 O exchange experiments reveal that the lineshapes of the broadband SFG spectra are significantly influenced by interference from OH oscillators in the aqueous phase, even when those oscillators are not probed by the incident infrared light in our broadband setup. In the absence of spectral interference from the OH stretches of the solvent, the alkyl chain terminal methyl group of the bilayer is found to be tilted at an angle of 15° to 35° from the surface normal.
Influence of membrane composition on its flexibility

International Nuclear Information System (INIS)

Gerbelli, B.B.; Teixeira da Silva, E.R.; Oliveira, C.L.P.; Oliveira, E.A.

2012-01-01

Full text: Lamellar phases and vesicles composed of lipids have been used as model systems to investigate biological process related to cell membrane as well as promising carriers for drugs and gene therapy. The composition of the membrane determines its three dimensional shape and its properties such as rigidity and compressibility which play an important role on membrane fusion, protein adhesion, interactions between proteins, etc. We present systematic study of a lamellar system composed mainly of lecithin which is a biocompatible phospholipid and simusol, which is a mixture of fatty acids that acts as a cosurfactant introducing flexibility to the membrane. Using X ray scattering we determine the lamellar periodicity as a function of the hydration for different formulations of the membrane; ranging from 100 % to 50 % mass fraction of lecithin. The X-ray spectra are fitted using a 4 Gaussian model [1]that allows us to determine the lamellar periodicity and the Caille parameter [2]. The ideal swelling law relating the membrane volume fraction (φ m ) to the lamellar periodicity (D) is given by φ m =δ m /D, where δ m is the thickness membrane, however, when steric interactions are dominant with respect to electrostatic and van der Waals interactions, deviations from this behavior are expected [3]. We present experimental data illustrating the swelling behavior for the membrane compositions and the respective behavior of the hydration limit, membrane Luzzati [4], of the Caille parameter and qualitative interpretation of the interaction forces the systems studying the parameter membrane square amplitude fluctuation[5]. [1] Private communication with Prof. Dr. Cristiano Luis Pinto de Oliveira. [2] Caille A. et all, Acad. Sci. Paris B274 (1972) 891. [3] E. Kurtisovski et all, PRL 98, 258103 (2007). [4] Nagle et all, Curr Opin Struct Biol. 2000 Aug;10(4):474-80. [5] H. I. Petrache. Structure and interactions of fluid phospholipids bilayers measured by high resolution
Influence of membrane composition on its flexibility

Energy Technology Data Exchange (ETDEWEB)

Gerbelli, B.B.; Teixeira da Silva, E.R.; Oliveira, C.L.P.; Oliveira, E.A. [Universidade de Sao Paulo (USP), SP (Brazil)

2012-07-01

Full text: Lamellar phases and vesicles composed of lipids have been used as model systems to investigate biological process related to cell membrane as well as promising carriers for drugs and gene therapy. The composition of the membrane determines its three dimensional shape and its properties such as rigidity and compressibility which play an important role on membrane fusion, protein adhesion, interactions between proteins, etc. We present systematic study of a lamellar system composed mainly of lecithin which is a biocompatible phospholipid and simusol, which is a mixture of fatty acids that acts as a cosurfactant introducing flexibility to the membrane. Using X ray scattering we determine the lamellar periodicity as a function of the hydration for different formulations of the membrane; ranging from 100 % to 50 % mass fraction of lecithin. The X-ray spectra are fitted using a 4 Gaussian model [1]that allows us to determine the lamellar periodicity and the Caille parameter [2]. The ideal swelling law relating the membrane volume fraction ({phi}{sub m}) to the lamellar periodicity (D) is given by {phi}{sub m} ={delta}{sub m}/D, where {delta}{sub m} is the thickness membrane, however, when steric interactions are dominant with respect to electrostatic and van der Waals interactions, deviations from this behavior are expected [3]. We present experimental data illustrating the swelling behavior for the membrane compositions and the respective behavior of the hydration limit, membrane Luzzati [4], of the Caille parameter and qualitative interpretation of the interaction forces the systems studying the parameter membrane square amplitude fluctuation[5]. [1] Private communication with Prof. Dr. Cristiano Luis Pinto de Oliveira. [2] Caille A. et all, Acad. Sci. Paris B274 (1972) 891. [3] E. Kurtisovski et all, PRL 98, 258103 (2007). [4] Nagle et all, Curr Opin Struct Biol. 2000 Aug;10(4):474-80. [5] H. I. Petrache. Structure and interactions of fluid phospholipids
Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Simon Imhof

2016-04-01

Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes. Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer. Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.
Progress in surface and membrane science

CERN Document Server

Cadenhead, D A

1976-01-01

Progress in Surface and Membrane Science, Volume 10 covers the advances in surface and membrane science. The book discusses the selective changes of cellular particles influencing sedimentation properties; and the rotating disk and ring-disk electrodes in investigations of surface phenomena at the metal-electrolyte interface. The text also describes the membrane potential of phospholipid bilayer and biological membranes; the adsorption of surfactant monolayers at gas/liquid and liquid/liquid interfaces; and the enzymes immobilized on glass. Chemists and people involved in electrochemistry will
Progress in surface and membrane science

CERN Document Server

Danielli, J F; Cadenhead, D A

1972-01-01

Progress in Surface and Membrane Science, Volume 5 covers the developments in the study of surface and membrane science. The book discusses the Mössbauer effect in surface science; the surface functional groups on carbon and silica; and the wetting phenomena pertaining to adhesion. The text also describes the physical state of phospholipids and cholesterol in monolayers, bilayers, and membranes; the characteristics of heterocoagulation; and the effects of calcium on excitable membranes and neurotransmitter action. Chemists, physiologists, biophysicists, and civil engineers will find the book i
Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Mote, Kaustubh R. [University of Minnesota, Department of Chemistry (United States); Gopinath, T. [University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics (United States); Veglia, Gianluigi, E-mail: vegli001@umn.edu [University of Minnesota, Department of Chemistry (United States)

2013-10-15

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD {approx}0.44 A, a tilt angle of 24 Degree-Sign {+-} 1 Degree-Sign , and an azimuthal angle of 55 Degree-Sign {+-} 6 Degree-Sign . This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.
ITO/Poly(Aniline/Sol-Gel Glass: An Optically Transparent, pH-Responsive Substrate for Supported Lipid Bilayers

Directory of Open Access Journals (Sweden)

Ahmed Al-Obeidi

2013-01-01

Full Text Available Described here is fabrication of a pH-sensitive, optically transparent transducer composed of a planar indium-tin oxide (ITO electrode overcoated with a poly(aniline (PANI thin film and a porous sol-gel layer. Adsorption of the PANI film renders the ITO electrode sensitive to pH, whereas the sol-gel spin-coated layer makes the upper surface compatible with fusion of phospholipid vesicles to form a planar supported lipid bilayer (PSLB. The response to changes in the pH of the buffer contacting the sol-gel/PANI/ITO electrode is pseudo-Nernstian with a slope of 52 mV/pH over a pH range of 4–9. Vesicle fusion forms a laterally continuous PSLB on the upper sol-gel surface that is fluid with a lateral lipid diffusion coefficient of 2.2 μm2/s measured by fluorescence recovery after photobleaching. Due to its lateral continuity and lack of defects, the PSLB blocks the pH response of the underlying electrode to changes in the pH of the overlying buffer. This architecture is simpler to fabricate than previously reported ITO electrodes derivatized for PSLB formation and should be useful for optical monitoring of proton transport across supported membranes derivatized with ionophores and ion channels.
Cell fusions in mammals

DEFF Research Database (Denmark)

Larsson, Lars-Inge; Bjerregaard, Bolette; Talts, Jan Fredrik

2008-01-01

Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear...... to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1......, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host ells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work...
Light-activated control of protein channel assembly mediated by membrane mechanics

Science.gov (United States)

Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

2016-12-01

Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.
Hydrophobic silver nanoparticles trapped in lipid bilayers: Size distribution, bilayer phase behavior, and optical properties

Directory of Open Access Journals (Sweden)

Bothun Geoffrey D

2008-11-01

Full Text Available Abstract Background Lipid-based dispersion of nanoparticles provides a biologically inspired route to designing therapeutic agents and a means of reducing nanoparticle toxicity. Little is currently known on how the presence of nanoparticles influences lipid vesicle stability and bilayer phase behavior. In this work, the formation of aqueous lipid/nanoparticle assemblies (LNAs consisting of hydrophobic silver-decanethiol particles (5.7 ± 1.8 nm embedded within 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC bilayers is demonstrated as a function of the DPPC/Ag nanoparticle (AgNP ratio. The effect of nanoparticle loading on the size distribution, bilayer phase behavior, and bilayer fluidity is determined. Concomitantly, the effect of bilayer incorporation on the optical properties of the AgNPs is also examined. Results The dispersions were stable at 50°C where the bilayers existed in a liquid crystalline state, but phase separated at 25°C where the bilayers were in a gel state, consistent with vesicle aggregation below the lipid melting temperature. Formation of bilayer-embedded nanoparticles was confirmed by differential scanning calorimetry and fluorescence anisotropy, where increasing nanoparticle concentration suppressed the lipid pretransition temperature, reduced the melting temperature, and disrupted gel phase bilayers. The characteristic surface plasmon resonance (SPR wavelength of the embedded nanoparticles was independent of the bilayer phase; however, the SPR absorbance was dependent on vesicle aggregation. Conclusion These results suggest that lipid bilayers can distort to accommodate large hydrophobic nanoparticles, relative to the thickness of the bilayer, and may provide insight into nanoparticle/biomembrane interactions and the design of multifunctional liposomal carriers.
Lipid bilayers and interfaces

NARCIS (Netherlands)

Kik, R.A.

2007-01-01

In biological systems lipid bilayers are subject to many different interactions with other entities. These can range from proteins that are attached to the hydrophilic region of the bilayer or transmembrane proteins that interact with the hydrophobic region of the lipid bilayer. Interaction between
Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

Science.gov (United States)

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

2009-08-13

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

DEFF Research Database (Denmark)

Skar-Gislinge, Nicholas

studies of membrane proteins. So far most of the studies using nanodiscs have been concerning the function of the incorporated membrane protein. However, due to the good control of the size and lipid composition of the nanodisc system, they seem an ideal tool for expanding the use of small angle......Phospholipid nanodiscs are ⇠ 10 nm disc shaped particles consisting of about 150 phospholipids arranged in a central bilayer stabilized by two amphipathic protein ”belts” that wrap around the rim of the bilayer. Because they contain a small bilayer leaflet they can be used as a tool for solution......-assembly process in general, and in particular in relation to incorporation of membrane proteins. This was the aim of work done early in this thesis. Here a detailed model for the small angle x-ray and neutron scattering from the empty nanodisc system was derived and used to describe the nanodisc system with great...

Deposition of phospholipid layers on SiO{sub 2} surface modified by alkyl-SAM islands

Energy Technology Data Exchange (ETDEWEB)

Tero, R.; Takizawa, M.; Li, Y.J.; Yamazaki, M.; Urisu, T

2004-11-15

Formation of the supported planar bilayer of dipalmitoylphosphatidylcholine (DPPC) on SiO{sub 2} surfaces modified with the self-assembled monolayer (SAM) of octadecyltrichlorosilane (OTS) has been investigated by atomic force microscopy (AFM). DPPC was deposited by the fusion of vesicles on SiO{sub 2} surfaces with OTS-SAM islands of different sizes and densities. The DPPC bilayer membrane formed self-organizingly on the SiO{sub 2} surface with small and sparse OTS islands, while did not when the OTS islands were larger and denser. The relative size between the vesicles and the SiO{sub 2} regions is the critical factor for the formation of the DPPC bilayer membrane.
Remotely controlled fusion of selected vesicles and living cells: a key issue review

Science.gov (United States)

Bahadori, Azra; Moreno-Pescador, Guillermo; Oddershede, Lene B.; Bendix, Poul M.

2018-03-01

Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.
Thermal and active fluctuations of a compressible bilayer vesicle

Science.gov (United States)

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

2018-05-01

We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.
SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.

Science.gov (United States)

Raghava, Smita; Giorda, Kristina M; Romano, Fabian B; Heuck, Alejandro P; Hebert, Daniel N

2013-06-04

Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1-5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.
H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion.

Science.gov (United States)

Baumann, Jan; Kouassi, Nancy Mounogou; Foni, Emanuela; Klenk, Hans-Dieter; Matrosovich, Mikhail

2016-02-01

The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T492S, N722D, R752K, and S1132F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased
Experimental approaches to membrane thermodynamics

DEFF Research Database (Denmark)

Westh, Peter

2009-01-01

Thermodynamics describes a system on the macroscopic scale, yet it is becoming an important tool for the elucidation of many specific molecular aspects of membrane properties. In this note we discuss this application of thermodynamics, and give a number of examples on how thermodynamic measurements...... have contributed to the understanding of specific membrane phenomena. We mainly focus on non-specific interactions of bilayers and small molecules (water and solutes) in the surrounding solvent, and the changes in membrane properties they bring about. Differences between thermodynamic...
Medium-chain fatty acid binding to albumin and transfer to phospholipid bilayers

International Nuclear Information System (INIS)

Hamilton, J.A.

1989-01-01

Temperature-dependent (5-42 degree C) 13 C NMR spectra of albumin complexes with 90% isotopically substituted [1- 13 C]octanoic or [1- 13 C]decanoic acids showed a single peak at >30 degree C but three peaks at lower temperatures. The chemical-shift differences result from different ionic and/or hydrogen-bonding interactions between amino acid side chains and the fatty acid carboxyl carbon. Rapid exchange of fatty acid among binding sites obscures these sites at temperatures >30 degree C. Rate constants for exchange at 33 degree C were 350 sec -1 for octanoate and 20 sec -1 for decanoate. Temperature-dependent data for octanoate showed an activation energy of 2 kcal/mol for exchange. Spectra of albumin complexes with the 12-carbon saturated fatty acid, lauric acid, had several narrow laurate carboxyl peaks at 35 degree C, indicating longer lifetimes in the different binding sites. Fatty acid exchange between albumin and model membranes (phosphatidylcholine bilayers) occurred on a time scale comparable to that for exchange among albumin binding sites, following the order octanoate > decanoate > laurate. The equilibrium distribution of fatty acid between lipid bilayers and protein was measured directly from NMR spectra. Decreasing pH increased the relative affinity of fatty acid for the lipid bilayer. The results predict that the relative affinity of octanoic acid for albumin and membranes will be similar to that of long-chain fatty acids, but the rate of equilibration will be ∼ 10 4 faster for octanoic acid
The Highly Conserved Proline at Position 438 in Pseudorabies Virus gH Is Important for Regulation of Membrane Fusion

OpenAIRE

Schröter, Christina; Klupp, Barbara G.; Fuchs, Walter; Gerhard, Marika; Backovic, Marija; Rey, Felix A.; Mettenleiter, Thomas C.

2014-01-01

Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure sho...
Nanodisc-solubilized membrane protein library reflects the membrane proteome.

Science.gov (United States)

Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

2013-05-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.
Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

Science.gov (United States)

Pohl, P; Saparov, S M; Borgnia, M J; Agre, P

2001-08-14

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.
Membrane alternatives in worlds without oxygen: Creation of an azotosome.

Science.gov (United States)

Stevenson, James; Lunine, Jonathan; Clancy, Paulette

2015-02-01

The lipid bilayer membrane, which is the foundation of life on Earth, is not viable outside of biology based on liquid water. This fact has caused astronomers who seek conditions suitable for life to search for exoplanets within the "habitable zone," the narrow band in which liquid water can exist. However, can cell membranes be created and function at temperatures far below those at which water is a liquid? We take a step toward answering this question by proposing a new type of membrane, composed of small organic nitrogen compounds, that is capable of forming and functioning in liquid methane at cryogenic temperatures. Using molecular simulations, we demonstrate that these membranes in cryogenic solvent have an elasticity equal to that of lipid bilayers in water at room temperature. As a proof of concept, we also demonstrate that stable cryogenic membranes could arise from compounds observed in the atmosphere of Saturn's moon, Titan, known for the existence of seas of liquid methane on its surface.
SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

International Nuclear Information System (INIS)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

2009-01-01

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.
Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

International Nuclear Information System (INIS)

Zambetti, G.; Stein, J.; Stein, G.

1987-01-01

The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression
Progress in surface and membrane science

CERN Document Server

Cadenhead, D A; Rosenberg, M D

1974-01-01

Progress in Surface and Membrane Science, Volume 8 covers the developments in the study of surface and membrane science. The book discusses the applications of statistical mechanics to physical adsorption; the impact of electron spectroscopy and cognate techniques on the study of solid surfaces; and the ellipsometric studies of thin films. The text also describes the interfacial photochemistry of bilayer lipid membranes; cell junctions and their development; and the composition and function of the inner mitochondrial membrane. The role of the cell surface in contact inhibition of cell division
Transmembrane Peptides as Sensors of the Membrane Physical State

Directory of Open Access Journals (Sweden)

Stefano Piotto

2018-05-01

Full Text Available Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state (MPS suggested a new approach for the discovery of antimicrobial peptides.
Comparative computational study of interaction of C60-fullerene and tris-malonyl-C60-fullerene isomers with lipid bilayer: relation to their antioxidant effect.

Directory of Open Access Journals (Sweden)

Marine E Bozdaganyan

Full Text Available Oxidative stress induced by excessive production of reactive oxygen species (ROS has been implicated in the etiology of many human diseases. It has been reported that fullerenes and some of their derivatives-carboxyfullerenes-exhibits a strong free radical scavenging capacity. The permeation of C60-fullerene and its amphiphilic derivatives-C3-tris-malonic-C60-fullerene (C3 and D3-tris-malonyl-C60-fullerene (D3-through a lipid bilayer mimicking the eukaryotic cell membrane was studied using molecular dynamics (MD simulations. The free energy profiles along the normal to the bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC for C60, C3 and D3 were calculated. We found that C60 molecules alone or in clusters spontaneously translocate to the hydrophobic core of the membrane and stay inside the bilayer during the whole period of simulation time. The incorporation of cluster of fullerenes inside the bilayer changes properties of the bilayer and leads to its deformation. In simulations of the tris-malonic fullerenes we discovered that both isomers, C3 and D3, adsorb at the surface of the bilayer but only C3 tends to be buried in the area of the lipid headgroups forming hydrophobic contacts with the lipid tails. We hypothesize that such position has implications for ROS scavenging mechanism in the specific cell compartments.
Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.

Science.gov (United States)

Barriga, Hanna M G; Booth, Paula; Haylock, Stuart; Bazin, Richard; Templer, Richard H; Ces, Oscar

2014-09-06

Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid-protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.
The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

Science.gov (United States)

Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

2007-07-15

Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.
Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface.

Science.gov (United States)

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

2016-03-01

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

Science.gov (United States)

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

2016-10-01

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

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