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Sample records for membranes preferential interaction

  1. C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake.

    PubMed

    Cordeiro Pedrosa, Lília R; van Cappellen, Wiggert A; Steurer, Barbara; Ciceri, Dalila; ten Hagen, Timo L M; Eggermont, Alexander M M; Verheij, Marcel; Goñi, Felix María; Koning, Gerben A; Contreras, F-Xabier

    2015-08-01

    Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox. SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.

  2. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  3. Steric effects and preferential interactions in supercritical carbon dioxide

    SciTech Connect

    Saquing, C.D.; Lucien, F.P.; Foster, N.R

    1998-10-01

    Solubility data are presented for a mixture of o-hydroxybenzoic acid (o-HBA) and m-HBA in supercritical CO{sub 2} doped with 3.5 mol% methanol. The data were measured at 318 and 328 K and for pressures in the range of 101--201 bar. Some new data for the solubility of pure m-HBA in methanol-doped supercritical CO{sub 2} are also presented. The solubilities of the HBA isomers are enhanced considerably with the addition of methanol to supercritical CO{sub 2}. However, the solubility enhancement is strongly affected by the spatial arrangement of their functional groups (steric effect). There appears to be preferential interaction between the solutes and the cosolvent in the quaternary system, and this phenomenon is consistent with thermodynamic modeling of the system.

  4. Membrane formation by preferential solvation of ions in mixture of water, 3-methylpyridine, and sodium tetraphenylborate

    SciTech Connect

    Sadakane, Koichiro; Nagao, Michihiro; Endo, Hitoshi; Seto, Hideki

    2013-12-21

    The structure and dynamics of a ternary system composed of deuterium oxide (D{sub 2}O), 3-methylpyridine (3MP), and sodium tetraphenylborate (NaBPh{sub 4}) are investigated by means of small-angle neutron scattering (SANS) and neutron spin echo (NSE) techniques. In the SANS experiments, a structural phase transition is confirmed between a disordered-phase and an ordered-lamellar-phase upon variation of the composition and/or temperature of the mixture. The characteristic lengths of the structures is on the sub-micrometer scale. A dispersion relation of the structure is measured through NSE experiments, which shows that the relaxation rate follows a cubic relation with momentum transfer. This implies that the dynamics of the system are determined predominantly by membrane fluctuations. The present results indicate that 3MP-rich domains are microscopically separated from bulk water in the presence of NaBPh{sub 4}, and that the layers behave as membranes. These results are interpreted that preferential solvation of salt in each solvent induces a microphase separation between the solvents, and the periodic structure of 3MP-rich domains is stabilized by the long-range electrostatic interaction arising from Na{sup +} ions in D{sub 2}O-rich domains.

  5. Its preferential interactions with biopolymers account for diverse observed effects of trehalose.

    PubMed

    Hong, Jiang; Gierasch, Lila M; Liu, Zhicheng

    2015-07-01

    Biopolymer homeostasis underlies the health of organisms, and protective osmolytes have emerged as one strategy used by Nature to preserve biopolymer homeostasis. However, a great deal remains unknown about the mechanism of action of osmolytes. Trehalose, as a prominent example, stabilizes proteins against denaturation by extreme temperature and denaturants, preserves membrane integrity upon freezing or in dry conditions, inhibits polyQ-mediated protein aggregation, and suppresses the aggregation of denatured proteins. The underlying thermodynamic mechanisms of such diverse effects of trehalose remain unclear or controversial. In this study, we applied the surface-additive method developed in the Record laboratory to attack this issue. We characterized the key features of trehalose-biopolymer preferential interactions and found that trehalose has strong unfavorable interactions with aliphatic carbon and significant favorable interactions with amide/anionic oxygen. This dissection has allowed us to elucidate the diverse effects of trehalose and to identify the crucial functional group(s) responsible for its effects. With (semi)quantitative thermodynamic analysis, we discovered that 1) the unfavorable interaction of trehalose with hydrophobic surfaces is the dominant factor in its effect on protein stability, 2) the favorable interaction of trehalose with polar amides enables it to inhibit polyQ-mediated protein aggregation and the aggregation of denatured protein in general, and 3) the favorable interaction of trehalose with phosphate oxygens, together with its unfavorable interaction with aliphatic carbons, enables trehalose to preserve membrane integrity in aqueous solution. These results provide a basis for a full understanding of the role of trehalose in biopolymer homeostasis and the reason behind its evolutionary selection as an osmolyte, as well as for a better application of trehalose as a chemical chaperone.

  6. Its Preferential Interactions with Biopolymers Account for Diverse Observed Effects of Trehalose

    PubMed Central

    Hong, Jiang; Gierasch, Lila M.; Liu, Zhicheng

    2015-01-01

    Biopolymer homeostasis underlies the health of organisms, and protective osmolytes have emerged as one strategy used by Nature to preserve biopolymer homeostasis. However, a great deal remains unknown about the mechanism of action of osmolytes. Trehalose, as a prominent example, stabilizes proteins against denaturation by extreme temperature and denaturants, preserves membrane integrity upon freezing or in dry conditions, inhibits polyQ-mediated protein aggregation, and suppresses the aggregation of denatured proteins. The underlying thermodynamic mechanisms of such diverse effects of trehalose remain unclear or controversial. In this study, we applied the surface-additive method developed in the Record laboratory to attack this issue. We characterized the key features of trehalose-biopolymer preferential interactions and found that trehalose has strong unfavorable interactions with aliphatic carbon and significant favorable interactions with amide/anionic oxygen. This dissection has allowed us to elucidate the diverse effects of trehalose and to identify the crucial functional group(s) responsible for its effects. With (semi)quantitative thermodynamic analysis, we discovered that 1) the unfavorable interaction of trehalose with hydrophobic surfaces is the dominant factor in its effect on protein stability, 2) the favorable interaction of trehalose with polar amides enables it to inhibit polyQ-mediated protein aggregation and the aggregation of denatured protein in general, and 3) the favorable interaction of trehalose with phosphate oxygens, together with its unfavorable interaction with aliphatic carbons, enables trehalose to preserve membrane integrity in aqueous solution. These results provide a basis for a full understanding of the role of trehalose in biopolymer homeostasis and the reason behind its evolutionary selection as an osmolyte, as well as for a better application of trehalose as a chemical chaperone. PMID:26153711

  7. Preferential Interaction of Na+ over K+ to Carboxylate-functionalized Silver Nanoparticles

    EPA Science Inventory

    Elucidating mechanistic interactions between specific ions (Na+/ K+) and nanoparticle surfaces to alter particle stability in polar media has received little attention. We investigated relative preferential binding of Na+ and K+ to carboxylate-functionalized silver nanoparticles ...

  8. Structure of the N-terminal segment of human retinol dehydrogenase 11 and its preferential lipid binding using model membranes.

    PubMed

    Lhor, Mustapha; Méthot, Mario; Horchani, Habib; Salesse, Christian

    2015-03-01

    Retinol dehydrogenase 11 (RDH11) has been postulated to be anchored to membranes by means of its N-terminal segment in retinal pigment epithelial (RPE) cells where it participates to the visual cycle. The analysis of the primary sequence of RDH11 revealed that its N-terminal hydrophobic segment could be involved in the anchoring of this enzyme to membranes. However, no information is yet available on the properties of this N-terminal segment to support this role. The secondary structure and membrane binding of two N-terminal peptides of RDH11 with different lengths have thus been investigated to provide this information. Online tools allowed predicting an α-helical secondary structure for both peptides. Infrared spectroscopy and circular dichroism have shown that the α-helix of the Long-peptide (35 amino acids) is longer and more rigid than that of the Short-peptide (25 amino acids) regardless of the type of solvent. Langmuir monolayers have been used as a model membrane to study lipid-peptide interactions. Values of maximum insertion pressure and synergy suggested a preferential binding of the Long-peptide to lipids with a phosphoethanolamine polar head group, which are abundant in the RPE. Furthermore, infrared spectroscopy in monolayers has shown that the α-helical structure of the Long-peptide is more stable in the presence of saturated phospholipids whereas the structure of the Short-peptide is mainly disordered. Altogether, the present data demonstrate that the α-helical hydrophobic core of the N-terminal segment of RDH11 displays properties typical of transmembrane domains, in agreement with its postulated role in the membrane anchoring of this protein.

  9. Multipronged interaction of the COG complex with intracellular membranes

    PubMed Central

    Willett, Rose; Pokrovskaya, Irina; Kudlyk, Tetyana; Lupashin, Vladimir

    2014-01-01

    The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion. PMID:24649395

  10. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  11. The interactions of peripheral membrane proteins with biological membranes.

    PubMed

    Whited, A M; Johs, A

    2015-11-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  12. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  13. Targeting LSCs through membrane antigens selectively or preferentially expressed on these cells.

    PubMed

    Pelosi, Elvira; Castelli, Germana; Testa, Ugo

    2015-12-01

    Studies of xenotransplantation of bone marrow and blood cells of AML patients have supported the existence of rare leukemic stem cells, able to initiate and maintain the leukemic process and bearing the typical leukemic abnormalities. LSCs possess self-renewal capacity and are responsible for the growth of the more differentiated leukemic progeny in the bone marrow and in the blood. These cells are more resistant than bulk leukemic cells to anti-leukemic drugs, thus survive to treatment and are, at a large extent, responsible for leukemia relapse. During the last two decades, considerable progresses have been made in the understanding of the peculiar cellular and molecular properties of LSCs. In this context, particularly relevant was the discovery of several membrane markers, selectively or preferentially expressed on LSCs. These membrane markers offer now unique opportunities to identify LSCs and to distinguish them from normal HSCs, to monitor the response of the various anti-leukemic treatments at the level of the LSC compartment, to identify relevant therapeutic targets. Concerning this last point, the most promising therapeutic targets are CD33 and CD123.

  14. Interaction of multiple biomimetic antimicrobial polymers with model bacterial membranes

    SciTech Connect

    Baul, Upayan Vemparala, Satyavani; Kuroda, Kenichi

    2014-08-28

    Using atomistic molecular dynamics simulations, interaction of multiple synthetic random copolymers based on methacrylates on prototypical bacterial membranes is investigated. The simulations show that the cationic polymers form a micellar aggregate in water phase and the aggregate, when interacting with the bacterial membrane, induces clustering of oppositely charged anionic lipid molecules to form clusters and enhances ordering of lipid chains. The model bacterial membrane, consequently, develops lateral inhomogeneity in membrane thickness profile compared to polymer-free system. The individual polymers in the aggregate are released into the bacterial membrane in a phased manner and the simulations suggest that the most probable location of the partitioned polymers is near the 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) clusters. The partitioned polymers preferentially adopt facially amphiphilic conformations at lipid-water interface, despite lacking intrinsic secondary structures such as α-helix or β-sheet found in naturally occurring antimicrobial peptides.

  15. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    PubMed

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography.

  16. Effective interactions between fluid membranes.

    PubMed

    Lu, Bing-Sui; Podgornik, Rudolf

    2015-08-01

    A self-consistent theory is proposed for the general problem of interacting undulating fluid membranes subject to the constraint that they do not interpenetrate. We implement the steric constraint via an exact functional integral representation and, through the use of a saddle-point approximation, transform it into a novel effective steric potential. The steric potential is found to consist of two contributions: one generated by zero-mode fluctuations of the membranes and the other by thermal bending fluctuations. For membranes of cross-sectional area S, we find that the bending fluctuation part scales with the intermembrane separation d as d-2 for d≪√S but crosses over to d-4 scaling for d≫√S, whereas the zero-mode part of the steric potential always scales as d-2. For membranes interacting exclusively via the steric potential, we obtain closed-form expressions for the effective interaction potential and for the rms undulation amplitude σ, which becomes small at low temperatures T and/or large bending stiffnesses κ. Moreover, σ scales as d for d≪√S but saturates at √kBTS/κ for d≫√S. In addition, using variational Gaussian theory, we apply our self-consistent treatment to study intermembrane interactions subject to different types of potentials: (i) the Moreira-Netz potential for a pair of strongly charged membranes with an intervening solution of multivalent counterions, (ii) an attractive square well, (iii) the Morse potential, and (iv) a combination of hydration and van der Waals interactions. PMID:26382349

  17. Effective interactions between fluid membranes

    NASA Astrophysics Data System (ADS)

    Lu, Bing-Sui; Podgornik, Rudolf

    2015-08-01

    A self-consistent theory is proposed for the general problem of interacting undulating fluid membranes subject to the constraint that they do not interpenetrate. We implement the steric constraint via an exact functional integral representation and, through the use of a saddle-point approximation, transform it into a novel effective steric potential. The steric potential is found to consist of two contributions: one generated by zero-mode fluctuations of the membranes and the other by thermal bending fluctuations. For membranes of cross-sectional area S , we find that the bending fluctuation part scales with the intermembrane separation d as d-2 for d ≪√{S } but crosses over to d-4 scaling for d ≫√{S } , whereas the zero-mode part of the steric potential always scales as d-2. For membranes interacting exclusively via the steric potential, we obtain closed-form expressions for the effective interaction potential and for the rms undulation amplitude σ , which becomes small at low temperatures T and/or large bending stiffnesses κ . Moreover, σ scales as d for d ≪√{S } but saturates at √{kBT S /κ } for d ≫√{S } . In addition, using variational Gaussian theory, we apply our self-consistent treatment to study intermembrane interactions subject to different types of potentials: (i) the Moreira-Netz potential for a pair of strongly charged membranes with an intervening solution of multivalent counterions, (ii) an attractive square well, (iii) the Morse potential, and (iv) a combination of hydration and van der Waals interactions.

  18. Interactions of amino acid side-chain analogs within membrane environments.

    PubMed

    Mirjalili, Vahid; Feig, Michael

    2015-02-19

    The interactions among four amino acid analog pairs (Asn, Ser, Phe, and Val) within the membrane environment were investigated using umbrella sampling molecular dynamics simulations. The results confirm generally expected qualitative trends of preferential association of polar compounds inside the membrane vs preferential interaction of hydrophobic compounds outside the membrane. Furthermore, correlations between amino acid interactions, membrane insertion, and membrane deformations are discussed and a detailed analysis of pair interaction energies is presented. A comparison of the energetics obtained from explicit lipid simulations with those from implicit membrane models reveals significant deviations and an improved parametrization of the heterogeneous dielectric generalized Born implicit model is provided that partially corrects for deficiencies in the implicit membrane model when compared with the new reference data from this study.

  19. Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella

    PubMed Central

    1991-01-01

    The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half- time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport. PMID:1874785

  20. Long hydrophilic-and-cationic polymers: a different pathway toward preferential activity against bacterial over mammalian membranes.

    PubMed

    Yang, Xin; Hu, Kan; Hu, Guantai; Shi, Danyao; Jiang, Yunjiang; Hui, Liwei; Zhu, Rui; Xie, Yuntao; Yang, Lihua

    2014-09-01

    We show that simply converting the hydrophobic moiety of an antimicrobial peptide (AMP) or synthetic mimic of AMPs (SMAMP) into a hydrophilic one could be a different pathway toward membrane-active antimicrobials preferentially acting against bacteria over host cells. Our biostatistical analysis on natural AMPs indicated that shorter AMPs tend to be more hydrophobic, and the hydrophilic-and-cationic mutants of a long AMP experimentally demonstrated certain membrane activity against bacteria. To isolate the effects of antimicrobials' hydrophobicity and systematically examine whether hydrophilic-and-cationic mutants could inherit the membrane activity of their parent AMPs/SMAMPs, we constructed a minimal prototypical system based on methacrylate-based polymer SMAMPs and compared the antibacterial membrane activity and hemolytic toxicity of analogues with and without the hydrophobic moiety. Antibacterial assays showed that the hydrophobic moiety of polymer SMAMPs consistently promoted the antibacterial activity but diminished in effectiveness for long polymers, and the resultant long hydrophilic-and-cationic polymers were also membrane active against bacteria. What distinguished these long mutants from their parent SMAMPs were their drastically reduced hemolytic toxicities and, as a result, strikingly enhanced selectivity. Similar toxicity reduction was observed with the hydrophilic-and-cationic mutants of long AMPs. Taken together, our results suggest that long hydrophilic-and-cationic polymers could offer preferential membrane activity against bacteria over host cells, which may have implications in future antimicrobial development.

  1. Membrane-mediated interactions measured using membrane domains.

    PubMed

    Semrau, Stefan; Idema, Timon; Schmidt, Thomas; Storm, Cornelis

    2009-06-17

    Cell membrane organization is the result of the collective effect of many driving forces. Several of these, such as electrostatic and van der Waals forces, have been identified and studied in detail. In this article, we investigate and quantify another force, the interaction between inclusions via deformations of the membrane shape. For electrically neutral systems, this interaction is the dominant organizing force. As a model system to study membrane-mediated interactions, we use phase-separated biomimetic vesicles that exhibit coexistence of liquid-ordered and liquid-disordered lipid domains. The membrane-mediated interactions between these domains lead to a rich variety of effects, including the creation of long-range order and the setting of a preferred domain size. Our findings also apply to the interaction of membrane protein patches, which induce similar membrane shape deformations and hence experience similar interactions.

  2. The organochlorine herbicide chloridazon interacts with cell membranes.

    PubMed

    Suwalsky, M; Benites, M; Villena, F; Norris, B; Quevedo, L

    1998-07-01

    Chloridazon is a widely used organochlorine herbicide. In order to evaluate its perturbing effect on cell membranes it was made to interact with human erythrocytes, frog adrenergic neuroepithelial synapse and molecular models. These consisted in multilayers of dimyristoylphosphatidylethanolamine (DMPE) and of dimyristoylphosphatidyltidylcholine (DMPC), representative of phospholipid classes located in the inner and outer monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that chloridazon interacted preferentially with DMPC multilayers. Scanning electron microscopy revealed that 0.1 mM chloridazon induced erythrocyte crenation. According to the bilayer couple hypothesis, this is due to the preferential insertion of chloridazon in the phosphatidylcholine-rich external moiety of the red cell membrane. Electrophysiological measurements showed that nerve stimulation was followed immediately by a transient increase in short-circuit current (SCC) and in the potential difference (PD) of the neuroepithelial synapse. Increasing concentrations of chloridazon caused a dose-dependent and reversible decrease of the responses of both parameters to 76% of their control values. The pesticide induced a similar (28%) significant time-dependent decrease in the basal values of the SCC and of PD. These results are in accordance with a perturbing effect of chloridazon on the phospholipid moiety of the nerve fibre membrane leading to interference with total ion transport across the nerve skin junction. PMID:9827013

  3. Pointlike Inclusion Interactions in Tubular Membranes

    NASA Astrophysics Data System (ADS)

    Vahid, Afshin; Idema, Timon

    2016-09-01

    Membrane tubes and tubular networks are ubiquitous in living cells. Inclusions like proteins are vital for both the stability and the dynamics of such networks. These inclusions interact via the curvature deformations they impose on the membrane. We analytically study the resulting membrane mediated interactions in strongly curved tubular membranes. We model inclusions as constraints coupled to the curvature tensor of the membrane tube. First, as special test cases, we analyze the interaction between ring- and rod-shaped inclusions. Using Monte Carlo simulations, we further show how pointlike inclusions interact to form linear aggregates. To minimize the curvature energy of the membrane, inclusions self-assemble into either line- or ringlike patterns. Our results show that the global curvature of the membrane strongly affects the interactions between proteins embedded in it, and can lead to the spontaneous formation of biologically relevant structures.

  4. CORE-Net: exploiting prior knowledge and preferential attachment to infer biological interaction networks.

    PubMed

    Montefusco, F; Cosentino, C; Amato, F

    2010-09-01

    The problem of reverse engineering in the topology of functional interaction networks from time-course experimental data has received considerable attention in literature, due to the potential applications in the most diverse fields, comprising engineering, biology, economics and social sciences. The present work introduces a novel technique, CORE-Net, which addresses this problem focusing on the case of biological interaction networks. The method is based on the representation of the network in the form of a dynamical system and on an iterative convex optimisation procedure. A first advantage of the proposed approach is that it allows to exploit qualitative prior knowledge about the network interactions, of the same kind as typically available from biological literature and databases. A second novel contribution consists of exploiting the growth and preferential attachment mechanisms to improve the inference performances when dealing with networks which exhibit a scale-free topology. The technique is first assessed through numerical tests on in silico random networks, subsequently it is applied to reverse engineering a cell cycle regulatory subnetwork in Saccharomyces cerevisiae from experimental microarray data. These tests show that the combined exploitation of prior knowledge and preferential attachment significantly improves the predictions with respect to other approaches.

  5. Biophysics of α-synuclein membrane interactions.

    PubMed

    Pfefferkorn, Candace M; Jiang, Zhiping; Lee, Jennifer C

    2012-02-01

    Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.

  6. Biophysics of α-Synuclein Membrane Interactions

    PubMed Central

    Pfefferkorn, Candace M.; Jiang, Zhiping; Lee, Jennifer C.

    2011-01-01

    Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson’s disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. PMID:21819966

  7. Interactions between Janus particles and membranes

    NASA Astrophysics Data System (ADS)

    Ding, Hong-Ming; Ma, Yu-Qiang

    2012-02-01

    Understanding how nanoparticles interact with cell membranes is of great importance in drug/gene delivery. In this paper, we investigate the interactions between Janus particles and membranes by using dissipative particle dynamics, and find that there exist two different modes (i.e., insertion and engulfment) in the Janus particle-membrane interactions. The initial orientation and properties of Janus particles have an important impact on the interactions. When the hydrophilic part of the particle is close to the membrane or the particle has a larger section area and higher hydrophilic coverage, the particle is more likely to be engulfed by the membrane. We also provide insights into the interactions between Janus particles and membranes containing lipid rafts, and find that a Janus particle could easily detach from a membrane after it is engulfed by the raft. The present study suggests a potential way to translocate Janus particles through membranes, which may give some significant suggestions on future nanoparticle design for drug delivery.

  8. Interaction of Defensins with Model Cell Membranes

    NASA Astrophysics Data System (ADS)

    Sanders, Lori K.; Schmidt, Nathan W.; Yang, Lihua; Mishra, Abhijit; Gordon, Vernita D.; Selsted, Michael E.; Wong, Gerard C. L.

    2009-03-01

    Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.

  9. Membrane-mediated interaction between retroviral capsids

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Nguyen, Toan

    2012-02-01

    A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.

  10. Preferential perpendicular acceleration of heavy ionospheric ions by interactions with electrostatic hydrogen cyclotron waves

    NASA Astrophysics Data System (ADS)

    Singh, N.; Schunk, R. W.; Sojka, J. J.

    1983-05-01

    Observations in recent years indicate the presence of energetic ions of ionospheric origin in various parts of the magnetosphere. These energetic ions have been found at all latitudes. Observations from the S3-3 satellite have made a great contribution toward an understanding of the energization of ionospheric ions. One of the most interesting observations is related to the finding that ion beams and electrostatic hydrogen cyclotron (EHC) waves are highly correlated and that they show an abrupt increase in their occurrence rate at an altitude of about 5000 km. A statistical survey of upward flowing ion (UFI) events occurring between 6000 and 8000 km has shown that the average energy of O(+) has a strong correlation with that of the H(+) ions. The present investigation has the objective to examine critically the energetics of UFI events in view of the theory of the interaction of a single coherent EHC wave with O(+), He(+), and H(+) ions. It is found that preferential acceleration of heavy ions occurs when such ions interact with an EHC wave.

  11. Preferential perpendicular acceleration of heavy ionospheric ions by interactions with electrostatic hydrogen cyclotron waves

    NASA Technical Reports Server (NTRS)

    Singh, N.; Schunk, R. W.; Sojka, J. J.

    1983-01-01

    Observations in recent years indicate the presence of energetic ions of ionospheric origin in various parts of the magnetosphere. These energetic ions have been found at all latitudes. Observations from the S3-3 satellite have made a great contribution toward an understanding of the energization of ionospheric ions. One of the most interesting observations is related to the finding that ion beams and electrostatic hydrogen cyclotron (EHC) waves are highly correlated and that they show an abrupt increase in their occurrence rate at an altitude of about 5000 km. A statistical survey of upward flowing ion (UFI) events occurring between 6000 and 8000 km has shown that the average energy of O(+) has a strong correlation with that of the H(+) ions. The present investigation has the objective to examine critically the energetics of UFI events in view of the theory of the interaction of a single coherent EHC wave with O(+), He(+), and H(+) ions. It is found that preferential acceleration of heavy ions occurs when such ions interact with an EHC wave.

  12. Preferential accumulation and enhanced relative velocity of inertial droplets due to interactions with homogeneous isotropic turbulence

    NASA Astrophysics Data System (ADS)

    Bateson, Colin; Aliseda, Alberto

    2015-11-01

    We present results from wind tunnel experiments on the evolution of small inertial (d ~ 10 - 200 μm) water droplets in homogeneous, isotropic, slowly decaying grid turbulence. High-speed imaging and a Particle Tracking algorithm are used to calculate relative velocity distributions. We analyze the preferential concentration, via the 2D Radial Distribution Function, and enhanced relative velocity of droplets resulting from their inertial interactions with the underlying turbulence. The two-dimensional particle velocities, measured from multi-image tracks along a streamwise plane, are conditionally analyzed with respect to the distance from the nearest particle. We focus on the non-normality of the statistics for the particle-particle separation velocity component to examine the influence of the inertial interaction with the turbulence on the dynamics of the droplets. We observe a negative bias (in the mean and mode) in the separation velocity of particles for short separations, signaling a tendency of particles to collide more frequently than a random agitation by turbulence would predict. The tails of the distribution are interpreted in terms of the collision/coalescence process and the probability of collisions that do not lead to coalescence.

  13. Interactions of chrysotile asbestos with erythrocyte membranes.

    PubMed

    Brody, A R; Hill, L H

    1983-09-01

    Chrysotile asbestos causes lysis of red blood cells. It has been proposed that the mechanism of hemolysis is mediated through interactions between asbestos and cell membrane glycoproteins. Our studies support this concept and the following results are reported. Electron microscopy shows that asbestos fibers distort red blood cells and bind to cell membranes which may become wrapped around the fibers. This reaction is prevented by pretreatment of the cells with neuraminidase. The distribution of lectins which bind to membrane glycoproteins is altered by treating the cells with asbestos. Cell distortion and membrane deformation consequent to asbestos treatment correlate with a clear increase in the ratio of intracellular Na+:K+ ions.

  14. Nanoparticles meet cell membranes: probing nonspecific interactions using model membranes.

    PubMed

    Chen, Kai Loon; Bothun, Geoffrey D

    2014-01-21

    Nanotoxicity studies have shown that both carbon-based and inorganic engineered nanoparticles can be toxic to microorganisms. Although the pathways for cytotoxicity are diverse and dependent upon the nature of the engineered nanoparticle and the chemical environment, numerous studies have provided evidence that direct contact between nanoparticles and bacterial cell membranes is necessary for cell inactivation or damage, and may in fact be a primary mechanism for cytotoxicity. The propensities for nanoparticles to attach to and disrupt cell membranes are still not well understood due to the heterogeneous and dynamic nature of biological membranes. Model biological membranes can be employed for systematic investigations of nanoparticle-membrane interactions. In this article, current and emerging experimental approaches to identify the key parameters that control the attachment of ENPs on model membranes and the disruption of membranes by ENPs will be discussed. This critical information will help enable the "safe-by-design" production of engineered nanoparticles that are nontoxic or biocompatible, and also allow for the design of antimicrobial nanoparticles for environmental and biomedical applications.

  15. Complete Switchgrass Genetic Maps Reveal Subgenome Collinearity, Preferential Pairing and Multilocus Interactions

    PubMed Central

    Okada, Miki; Lanzatella, Christina; Saha, Malay C.; Bouton, Joe; Wu, Rongling; Tobias, Christian M.

    2010-01-01

    Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages. PMID:20407132

  16. Cobra cardiotoxins: membrane interactions and pharmacological potential.

    PubMed

    Dubovskii, P V; Konshina, A G; Efremov, R G

    2014-01-01

    Natural polycationic membrane-active peptides typically lack disulfide bonds and exhibit fusion, cell-penetrating, antimicrobial activities. They are mostly unordered in solution, but adopt a helical structure, when bound to phospholipid membranes. Structurally different are cardiotoxins (or cytotoxins, CTs) from cobra venom. They are fully β- structured molecules, characterized by the three-finger fold (TFF). Affinity of CTs to lipid bilayer was shown to depend on amino acid sequence in the tips of the three loops. In the present review, CT-membrane interactions are analyzed through the prism of data on binding of the toxins to phospholipid liposomes and detergent micelles, as well as their structural and computational studies in membrane mimicking environments. We assess different hydrophobicity scales to compare membrane partitioning of various CTs and their membrane effects. A comparison of hydrophobic/hydrophilic properties of CTs and linear polycationic peptides provides a key to their biological activity and creates a fundamental basis for rational design of new membrane-interacting compounds, including new promising drugs. For instance, from the viewpoint of the data obtained on model lipid membranes, cytotoxic activity of CTs against cancer cells is discussed.

  17. Interaction of detergent sclerosants with cell membranes.

    PubMed

    Parsi, Kurosh

    2015-06-01

    Commonly used detergent sclerosants including sodium tetradecyl sulphate (STS) and polidocanol (POL) are clinically used to induce endovascular fibrosis and vessel occlusion. They achieve this by lysing the endothelial lining of target vessels. These agents are surface active (surfactant) molecules that interfere with cell membranes. Surfactants have a striking similarity to the phospholipid molecules of the membrane lipid bilayer. By adsorbing at the cell membrane, surfactants disrupt the normal architecture of the lipid bilayer and reduce the surface tension. The outcome of this interaction is concentration dependent. At high enough concentrations, surfactants solubilise cell membranes resulting in cell lysis. At lower concentrations, these agents can induce a procoagulant negatively charged surface on the external aspect of the cell membrane. The interaction is also influenced by the ionic charge, molecular structure, pH and the chemical nature of the diluent (e.g. saline vs. water). The ionic charge of the surfactant molecule can influence the effect on plasma proteins and the protein contents of cell membranes. STS, an anionic detergent, denatures the tertiary complex of most proteins and in particular the clinically relevant clotting factors. By contrast, POL has no effect on proteins due to its non-ionic structure. These agents therefore exhibit remarkable differences in their interaction with lipid membranes, target cells and circulating proteins with potential implications in a range of clinical applications.

  18. Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility.

    PubMed

    Shulgin, Ivan L; Ruckenstein, Eli

    2005-12-01

    The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.

  19. Local anesthetics structure-dependently interact with anionic phospholipid membranes to modify the fluidity.

    PubMed

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

    2010-01-01

    While bupivacaine is more cardiotoxic than other local anesthetics, the mechanistic background for different toxic effects remains unclear. Several cardiotoxic compounds act on lipid bilayers to change the physicochemical properties of membranes. We comparatively studied the interaction of local anesthetics with lipid membranous systems which might be related to their structure-selective cardiotoxicity. Amide local anesthetics (10-300 microM) were reacted with unilamellar vesicles which were prepared with different phospholipids and cholesterol of varying lipid compositions. They were compared on the potencies to modify membrane fluidity by measuring fluorescence polarization. Local anesthetics interacted with liposomal membranes to increase the fluidity. Increasing anionic phospholipids in membranes enhanced the membrane-fluidizing effects of local anesthetics with the potency being cardiolipin>phosphatidic acid>phosphatidylglycerol>phosphatidylserine. Cardiolipin was most effective on bupivacaine, followed by ropivacaine. Local anesthetics interacted differently with biomimetic membranes consisting of 10mol% cardiolipin, 50mol% other phospholipids and 40mol% cholesterol with the potency being bupivacaine>ropivacaine>lidocaine>prilocaine, which agreed with the rank order of cardiotoxicity. Bupivacaine significantly fluidized 2.5-12.5mol% cardiolipin-containing membranes at cardiotoxicologically relevant concentrations. Bupivacaine is considered to affect lipid bilayers by interacting electrostatically with negatively charged cardiolipin head groups and hydrophobically with phospholipid acyl chains. The structure-dependent interaction with lipid membranes containing cardiolipin, which is preferentially localized in cardiomyocyte mitochondrial membranes, may be a mechanistic clue to explain the structure-selective cardiotoxicity of local anesthetics.

  20. Islet amyloid polypeptide toxicity and membrane interactions.

    PubMed

    Cao, Ping; Abedini, Andisheh; Wang, Hui; Tu, Ling-Hsien; Zhang, Xiaoxue; Schmidt, Ann Marie; Raleigh, Daniel P

    2013-11-26

    Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of β-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity. PMID:24218607

  1. STARD4 Membrane Interactions and Sterol Binding.

    PubMed

    Iaea, David B; Dikiy, Igor; Kiburu, Irene; Eliezer, David; Maxfield, Frederick R

    2015-08-01

    The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix.

  2. Bilayer membrane interactions with nanofabricated scaffolds

    SciTech Connect

    Collier, C. Patrick

    2015-07-29

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

  3. Bilayer membrane interactions with nanofabricated scaffolds

    DOE PAGES

    Collier, C. Patrick

    2015-07-29

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

  4. Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity.

    PubMed

    Di Pasquale, Eric; Salmi-Smail, Chanaz; Brunel, Jean-Michel; Sanchez, Patrick; Fantini, Jacques; Maresca, Marc

    2010-02-01

    The interaction of squalamine (SQ) with eukaryotic and prokaryotic membranes was studied and compared with the interaction of two other cationic amphipathic antimicrobials (CAAs), i.e. the antibiotic polymyxin B (PMB) and the detergent hexadecyltrimethylammonium bromide (CTAB). Whole cell experiments showed that the three CAA have in common the ability to interact with lipopolysaccharide-containing membranes through a divalent cation sensitive process. Differences were found regarding their kinetics of membrane permeabilisation and their selectivity for bacteria, with a preferential permeabilisation of bacteria by PMB>SQ and no selectivity for CTAB. Experiments with lipid monolayers and bilayers showed that this selectivity did not correlate with a preferential interaction of the CAAs with lipids but rather relies on differences in their ability to penetrate lipid bilayers and to cause electrically active lesions. Incidentally, our results also suggest that the distribution coefficient of CAAs could be used to predict their selectivity for bacteria. PMID:19883637

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  6. Cu2+ ions interact with cell membranes.

    PubMed

    Suwalsky, M; Ungerer, B; Quevedo, L; Aguilar, F; Sotomayor, C P

    1998-07-01

    The influence of Cu2+ ions on the physical properties of resealed human erythrocyte membranes was studied by fluorescence spectroscopy. A net ordering effect was observed at the hydrophobic-hydrophilic interface both in the bulk as well as in the lipid-protein boundary. The explanation for this result was found by X-ray diffraction performed in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Cu2+ did not significantly affect the structure of DMPE; however, DMPC polar head and hydrocarbon chain arrangements were perturbed at low but reordered at high Cu2+ concentrations. These effects were respectively explained in terms of a limited and extended interaction between Cu2+ ions and DMPC PO4 groups. Thus, the ordering effect in the erythrocyte membrane could be based on the interaction of this cation with phosphatidylcholine phosphate groups located in its outer leaflet. This binding, besides producing a decrease of membrane fluidity, might also induce a change in its electric field. These two effects should affect the activity of membrane proteins, particularly of ion channels. In fact, it was found that increasing concentrations of Cu2+ ions applied to either the mucosal or serosal surface of the isolated toad skin elicited a dose-dependent decrease of the short-circuit current (SCC) and of the potential difference (PD). These results lead to the conclusion that Cu2+ ions inhibited Na+ transport across the epithelial cell membranes. PMID:9720309

  7. Functionalized nanoparticle interactions with polymeric membranes

    PubMed Central

    Ladner, D.A.; Steele, M.; Weir, A.; Hristovski, K.; Westerhoff, P.

    2011-01-01

    A series of experiments was performed to measure the retention of a class of functionalized nanoparticles (NPs) onporous (microfiltration and ultrafiltration) membranes. The findings impact engineered water and wastewater treatment using membrane technology, characterization and analytical schemes for NP detection, and the use of NPs in waste treatment scenarios. The NPs studied were composed of silver, titanium dioxide, and gold; had organic coatings to yield either positive or negative surface charge; and were between 2 and 10 nm in diameter. NP solutions were applied to polymeric membranes composed of different materials and pore sizes (ranging from ~2 nm [3 kDa molecular weight cutoff] to 0.2 μm). Greater than 99% rejection was observed of positively charged NPs by negatively charged membranes even though pore diameters were up to 20 times the NP diameter; thus, sorption caused rejection. Negatively charged NPs were less well rejected, but behavior was dependant not only on surface functionality but on NP core material (Ag, TiO2, or Au). NP rejection depended more upon NP properties than membrane properties; all of the negatively charged polymeric membranes behaved similarly. The NP-membrane interaction behavior fell into four categories, which are defined and described here. PMID:22177020

  8. Membrane–drug interactions studied using model membrane systems

    PubMed Central

    Knobloch, Jacqueline; Suhendro, Daniel K.; Zieleniecki, Julius L.; Shapter, Joseph G.; Köper, Ingo

    2015-01-01

    The direct interaction of drugs with the cell membrane is often neglected when drug effects are studied. Systematic investigations are hindered by the complexity of the natural membrane and model membrane systems can offer a useful alternative. Here some examples are reviewed of how model membrane architectures including vesicles, Langmuir monolayers and solid supported membranes can be used to investigate the effects of drug molecules on the membrane structure, and how these interactions can translate into effects on embedded membrane proteins. PMID:26586998

  9. Preferential interaction of the Alzheimer peptide Aβ-(1-42) with Omega-3-containing lipid bilayers: structure and interaction studies.

    PubMed

    Emendato, Alessandro; Spadaccini, Roberta; De Santis, Augusta; Guerrini, Remo; D'Errico, Gerardino; Picone, Delia

    2016-02-01

    Many age-related neurodegenerative diseases, including Alzheimer Disease (AD), are elicited by an interplay of genetic, environmental, and dietary factors. Food rich in Omega-3 phospholipids seems to reduce the AD incidence. To investigate the molecular basis of this beneficial effect, we have investigated by CD and ESR studies the interaction between the Alzheimer peptide Aβ-(1-42) and biomimetic lipid bilayers. The inclusion of 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine does not change significantly the bilayers organization, but favors its Aβ-(1-42) interaction. The Omega-3 lipid amount modulates the effect intensity, suggesting a peptide selectivity for membranes containing polyunsatured fatty acids (PUFA) and providing hints for the mechanism and therapy of AD. PMID:26821608

  10. Interaction of antiarrhythmic drugs with model membranes.

    PubMed

    Suwalsky, M; Sánchez, I; Bagnara, M; Sotomayor, C P

    1994-11-01

    Several hypotheses link the molecular mechanism of action of the antiarrhythmic drugs (AAD) that belong to class I to nonspecific interactions with phospholipids sited in the neighborhood of the sodium channels in the membrane of the myocard. The interactions of asocainol (ASOC), procainamide (PROC) and quinidine (QUIN) with: (a) multibilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), in both a hydrophobic and a hydrophilic medium, and (b) DMPC vesicles, were studied, respectively, by X-ray diffraction and fluorescence spectroscopy. It was found that the three AAD interacted with the lipid bilayers. However, the extension of these interactions depended on the nature and concentration of the lipids and AAD as well as on the medium where the interactions were performed. The different capacity of ASOC and PROC to perturb the bilayer structures, mainly that of DMPC, indicated that the interactions were strongly dependent on the lipophilicity of these drugs. The fact that QUIN did not completely interact in accordance to its lipophilicity suggested that other factors also play a role in these interactions. It is concluded that it may be valid the suggested molecular mechanisms of action of class I AAD involving their interaction with the membrane phospholipids. PMID:7947909

  11. Model membrane/substrate interactions: ethanol and procaine interactions.

    PubMed

    Cadenhead, D A.

    2001-09-01

    The ability of ethanol to lower the surface tension of water plays a major role in its ability to affect membrane lipids. Typical lipids will show little expansion on exposure to ethanol substrates and may even show condensations. At the same time, the overall stability of the lipid phase is significantly reduced. Previously, we reported experimental and theoretical studies of the stearic acid (SA)/procaine (PR) system. PR substrate concentrations were examined in the 10(-4)-10(-2) M range as Gibbs monolayers, in order to establish the surface activity of both charged and uncharged species, and to estimate the orientation of the PR species at the air/water interface. SA interactions with PR substrates were studied by compressing films of the former and recording the surface pressure/area per molecule isotherms at both pH 2 and 8, so that the SA was in an uncharged and charged state, respectively. More recently, we have carried out similar studies with L-alpha-dipalmitoyl phosphatidylcholine (DPPC), maintaining the substrate pH at between 5 and 6, at PR concentrations of 10(-6)-10(-2)M. We also carried out studies of the DPPC/PR system using fluorescence microscopy in order to examine the effects of PR on the biphasic liquid expanded/liquid condensed (LE/LC) transition region. In the absence of any lipid film, PR species appear to be horizontally oriented at the air/water interface, while the surface activity of PR species increases in the order PRH(2+)

  12. Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4.

    PubMed

    Huang, H; Schroeder, F; Zeng, C; Estes, M K; Schoer, J K; Ball, J M

    2001-04-01

    The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also plays a role in the unique rotavirus morphogenesis that involves a transient budding of newly made immature viral particles into the endoplasmic reticulum. NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NSP4(114-135)) mobilize intracellular calcium and induce secretory chloride currents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a lipid-binding domain are unresolved. Therefore, circular dichroism was used to determine (i) the interaction(s) of NSP4 and NSP4(114-135) with model membranes, (ii) the conformational changes elicited in NSP4 upon interacting with membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and (iv) the molar dissociation constant (K(d)) of NSP4(114-135) with defined lipid vesicles. Circular dichroism revealed for the first time that NSP4 and NSP4(114-135) undergo secondary structural changes upon interaction with membrane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) preferentially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV). Binding to SUV, but not LUV, was greatly enhanced by negatively charged phospholipids. Increasing the SUV cholesterol content, concomitant with the presence of negatively charged phospholipids, further potentiated the interaction of NSP4(114-135) with the SUV membrane. The K(d) of NSP4(114-135) was determined as well as partitioning of NSP4(114-135) with SUVs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae.

  13. Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4.

    PubMed

    Huang, H; Schroeder, F; Zeng, C; Estes, M K; Schoer, J K; Ball, J M

    2001-04-01

    The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also plays a role in the unique rotavirus morphogenesis that involves a transient budding of newly made immature viral particles into the endoplasmic reticulum. NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NSP4(114-135)) mobilize intracellular calcium and induce secretory chloride currents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a lipid-binding domain are unresolved. Therefore, circular dichroism was used to determine (i) the interaction(s) of NSP4 and NSP4(114-135) with model membranes, (ii) the conformational changes elicited in NSP4 upon interacting with membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and (iv) the molar dissociation constant (K(d)) of NSP4(114-135) with defined lipid vesicles. Circular dichroism revealed for the first time that NSP4 and NSP4(114-135) undergo secondary structural changes upon interaction with membrane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) preferentially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV). Binding to SUV, but not LUV, was greatly enhanced by negatively charged phospholipids. Increasing the SUV cholesterol content, concomitant with the presence of negatively charged phospholipids, further potentiated the interaction of NSP4(114-135) with the SUV membrane. The K(d) of NSP4(114-135) was determined as well as partitioning of NSP4(114-135) with SUVs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae. PMID:11300798

  14. Mutual Interactions between Aquaporins and Membrane Components

    PubMed Central

    Martínez-Ballesta, Maria del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein–protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances. PMID:27625676

  15. Mutual Interactions between Aquaporins and Membrane Components.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein-protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances.

  16. Mutual Interactions between Aquaporins and Membrane Components.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein-protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances. PMID:27625676

  17. Mutual Interactions between Aquaporins and Membrane Components

    PubMed Central

    Martínez-Ballesta, Maria del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein–protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances.

  18. Interaction of peptides with cell membranes: insights from molecular modeling

    NASA Astrophysics Data System (ADS)

    Li, Zhen-lu; Ding, Hong-ming; Ma, Yu-qiang

    2016-03-01

    The investigation of the interaction of peptides with cell membranes is the focus of active research. It can enhance the understanding of basic membrane functions such as membrane transport, fusion, and signaling processes, and it may shed light on potential applications of peptides in biomedicine. In this review, we will present current advances in computational studies on the interaction of different types of peptides with the cell membrane. Depending on the properties of the peptide, membrane, and external environment, the peptide-membrane interaction shows a variety of different forms. Here, on the basis of recent computational progress, we will discuss how different peptides could initiate membrane pores, translocate across the membrane, induce membrane endocytosis, produce membrane curvature, form fibrils on the membrane surface, as well as interact with functional membrane proteins. Finally, we will present a conclusion summarizing recent progress and providing some specific insights into future developments in this field.

  19. Interaction of Mastoparan with Model Membranes

    NASA Astrophysics Data System (ADS)

    Haloot, Justin

    2010-10-01

    The use of antimicrobial agents began during the 20th century to reduce the effects of infectious diseases. Since the 1990s, antimicrobial resistance has become an ever-increasing global problem. Our laboratory recently found that small antimicrobial peptides (AMPs) have potent antimicrobial activity against a wide range of Gram-negative and Gram-positive organisms including antibiotic resistant organisms. These AMPs are potential therapeutic agents against the growing problem of antimicrobial resistance. AMPs are small peptides produced by plants, insects and animals. Several hypotheses concede that these peptides cause some type of structural perturbations and increased membrane permeability in bacteria however, how AMPs kill bacteria remains unclear. The goal of this study was to design an assay that would allow us to evaluate and monitor the pore forming ability of an AMP, Mastoparan, on model membrane structures called liposomes. Development of this model will facilitate the study of how mastoparan and related AMPs interact with the bacterial membrane.

  20. Isoniazid interaction with phosphatidylcholine-based membranes

    NASA Astrophysics Data System (ADS)

    Marques, Amanda Vicente; Marengo Trindade, Paulo; Marques, Sheylla; Brum, Tainá; Harte, Etienne; Rodrigues, Marieli Oliveira; D'Oca, Marcelo Gonçalves Montes; da Silva, Pedro Almeida; Pohlmann, Adriana R.; Alves, Isabel Dantas; de Lima, Vânia Rodrigues

    2013-11-01

    Interaction between the anti-tuberculosis drug isoniazid (INH) and phosphatidylcholine membranes was investigated in terms of: (i) drug affinity to a lipid bilayer and (ii) drug-induced changes in the dynamic properties of liposomes, such as membrane hydration state, polar head and non-polar acyl chain order and lipid phase transition behavior. These parameters were studied by plasmon waveguide resonance spectroscopy (PWR), UV-visible, horizontal attenuated total reflectance-Fourier transform infrared (HATR-FTIR), nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) techniques. PWR measurements showed an INH membrane dissociation constant value of 0.031 μM to phosphatidylcholine bilayers. INH induced higher membrane perturbation in the plane which is perpendicular to the membrane plane. The INH saturation concentration in phosphatidylcholine liposomes was 170 μM. At this concentration, HATR-FTIR and NMR findings showed that INH may interact with the lipid polar head, increasing the number of hydrogen bonds in the phosphate region and enhancing the choline motional freedom. DSC measurements showed that, at 115 μM, INH was responsible for a decrease in lipid phase transition temperature of approximately 2 °C and had no influence in the lipid enthalpy variation (ΔH). However, at 170 μM, INH induced the reduction of the ΔH by approximately 52%, suggesting that the drug may increase the distance among lipid molecules and enhance the freedom of the lipid acyl chains methylene groups. This paper provides information on the effects of INH on membrane dynamics which is important to understand liposome targeting of the drug and for the development of anti-TB pharmacologic systems that not only are less susceptible to resistance but also have low toxicity.

  1. Interactions of Model Cell Membranes with Nanoparticles

    NASA Astrophysics Data System (ADS)

    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

  2. Curvature-mediated interactions between membrane proteins.

    PubMed Central

    Kim, K S; Neu, J; Oster, G

    1998-01-01

    Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions. PMID:9788923

  3. Toxins and antimicrobial peptides: interactions with membranes

    NASA Astrophysics Data System (ADS)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

  4. Cell-penetrating compounds preferentially bind glycosaminoglycans over plasma membrane lipids in a charge density- and stereochemistry-dependent manner.

    PubMed

    Prevette, Lisa E; Benish, Nicolas C; Schoenecker, Amber R; Braden, Kristin J

    2015-12-01

    Cell-penetrating compounds (CPCs) are often conjugated to drugs and genes to facilitate cellular uptake. We hypothesize that the electrostatic interaction between the positively charged amines of the cell-penetrating compounds and the negatively charged glycosaminoglycans (GAGs) extending from cell surfaces is the initiating step in the internalization process. The interactions of generation 5 PAMAM dendrimer, Tat peptide and 25 kDa linear PEI with four different GAGs have been studied using isothermal titration calorimetry to elucidate structure-function relationships that could lead to improved drug and gene delivery methods to a wide variety of cell types. Detailed thermodynamic analysis has determined that CPC-GAG binding constants range from 8.7×10(3) to 2.4×10(6)M(-1) and that affinity is dependent upon GAG charge density and stereochemistry and CPC molecular weight. The effect of GAG composition on affinity is likely due to hydrogen bonding between CPC amines and amides and GAG hydroxyl and amine groups. These results were compared to the association of CPCs with lipid vesicles of varying composition as model plasma membranes to finally clarify the relative importance of each cell surface component in initial cell recognition. CPC-lipid affinity increases with anionic lipid content, but GAG affinity is higher for all cell-penetrating compounds, confirming the role these heterogeneous polysaccharides play in cellular association and clustering.

  5. Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

    NASA Astrophysics Data System (ADS)

    Jones, Emmalee M.

    A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped

  6. Membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides.

    PubMed

    Braun, Katharina; Pochert, Alexander; Lindén, Mika; Davoudi, Mina; Schmidtchen, Artur; Nordström, Randi; Malmsten, Martin

    2016-08-01

    Membrane interactions are critical for the successful use of mesoporous silica nanoparticles as delivery systems for antimicrobial peptides (AMPs). In order to elucidate these, we here investigate effects of nanoparticle charge and porosity on AMP loading and release, as well as consequences of this for membrane interactions and antimicrobial effects. Anionic mesoporous silica particles were found to incorporate considerable amounts of the cationic AMP LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37), whereas loading is much lower for non-porous or positively charged silica nanoparticles. Due to preferential pore localization, anionic mesoporous particles, but not the other particles, protect LL-37 from degradation by infection-related proteases. For anionic mesoporous nanoparticles, membrane disruption is mediated almost exclusively by peptide release. In contrast, non-porous silica particles build up a resilient LL-37 surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. For positively charged mesoporous silica nanoparticles, LL-37 incorporation promotes the membrane binding and disruption displayed by the particles in the absence of peptide, but also causes toxicity against human erythrocytes. Thus, the use of mesoporous silica nanoparticles as AMP delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nanoparticles, the latter critically dependent on nanoparticle properties. PMID:27174622

  7. Membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides.

    PubMed

    Braun, Katharina; Pochert, Alexander; Lindén, Mika; Davoudi, Mina; Schmidtchen, Artur; Nordström, Randi; Malmsten, Martin

    2016-08-01

    Membrane interactions are critical for the successful use of mesoporous silica nanoparticles as delivery systems for antimicrobial peptides (AMPs). In order to elucidate these, we here investigate effects of nanoparticle charge and porosity on AMP loading and release, as well as consequences of this for membrane interactions and antimicrobial effects. Anionic mesoporous silica particles were found to incorporate considerable amounts of the cationic AMP LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37), whereas loading is much lower for non-porous or positively charged silica nanoparticles. Due to preferential pore localization, anionic mesoporous particles, but not the other particles, protect LL-37 from degradation by infection-related proteases. For anionic mesoporous nanoparticles, membrane disruption is mediated almost exclusively by peptide release. In contrast, non-porous silica particles build up a resilient LL-37 surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. For positively charged mesoporous silica nanoparticles, LL-37 incorporation promotes the membrane binding and disruption displayed by the particles in the absence of peptide, but also causes toxicity against human erythrocytes. Thus, the use of mesoporous silica nanoparticles as AMP delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nanoparticles, the latter critically dependent on nanoparticle properties.

  8. Interaction of the N-terminus of sterol carrier protein 2 with membranes: role of membrane curvature.

    PubMed Central

    Huang, H; Ball, J M; Billheimer, J T; Schroeder, F

    1999-01-01

    Although neither the physiological function nor the mechanism of action of sterol carrier protein 2 (SCP(2)) is yet completely clear, it is thought that SCP(2) interacts with membranes to elicit its biological effects. The results presented here show that the SCP(2) N-terminus, composed of two amphipathic alpha-helices, interacted preferentially with highly curved but not lower-curvature membranes containing anionic phospholipid. CD spectra of SCP(2) showed up to 1. 2-fold increased alpha-helical content, on the interaction of SCP(2) with small unilamellar vesicles (SUV) (median radius 10-14 nm) but less with large unilamellar vesicles (LUV) (median radius 52-60 nm). Although enhanced interaction with the SUV membranes was due in part to the radius of curvature and to the greater exposure of acidic phospholipid in the outer leaflet of the bilayer, simply increasing the molar percentage of acidic phospholipid in the LUV membranes had much less effect on SCP(2) binding. A similar preferential interaction was observed with highly curved SUV as opposed to LUV for the SCP(2) N-terminal peptide (1-32)SCP(2) as well as structurally modified peptides in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>>(1-E20-32)SCP(2). The CD results were confirmed with an independent filtration binding assay, which showed that SCP(2) bound 5-fold more to SUV than LUV, whereas its N-terminal peptides bound up to 4-fold better in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>(1-E20-32)SCP(2). Finally, cholesterol potentiated the binding of SCP(2) and N-terminal peptides to anionic-phospholipid-containing SUV but not LUV. These findings were consistent with the SCP(2) N-terminus being a membrane-binding domain that was highly dependent on membrane surface curvature as well as on lipid composition. PMID:10567245

  9. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  10. Molecular mechanisms of membrane interaction at implantation.

    PubMed

    Davidson, Lien M; Coward, Kevin

    2016-03-01

    Successful pregnancy is dependent upon the implantation of a competent embryo into a receptive endometrium. Despite major advancement in our understanding of reproductive medicine over the last few decades, implantation failure still occurs in both normal pregnancies and those created artificially by assisted reproductive technology (ART). Consequently, there is significant interest in elucidating the etiology of implantation failure. The complex multistep process of implantation begins when the developing embryo first makes contact with the plasma membrane of epithelial cells within the uterine environment. However, although this biological interaction marks the beginning of a fundamental developmental process, our knowledge of the intricate physiological and molecular processes involved remains sparse. In this synopsis, we aim to provide an overview of our current understanding of the morphological changes which occur to the plasma membrane of the uterine endothelium, and the molecular mechanisms that control communication between the early embryo and the endometrium during implantation. A multitude of molecular factors have been implicated in this complex process, including endometrial integrins, extracellular matrix molecules, adhesion molecules, growth factors, and ion channels. We also explore the development of in vitro models for embryo implantation to help researchers investigate mechanisms which may underlie implantation failure. Understanding the precise molecular pathways associated with implantation failure could help us to generate new prognostic/diagnostic biomarkers, and may identify novel therapeutic targets. PMID:26969610

  11. Separation of preferential interaction and excluded volume effects on DNA duplex and hairpin stability

    PubMed Central

    Knowles, D. B.; LaCroix, Andrew S.; Deines, Nickolas F.; Shkel, Irina; Record, M. Thomas

    2011-01-01

    Small solutes affect protein and nucleic acid processes because of favorable or unfavorable chemical interactions of the solute with the biopolymer surface exposed or buried in the process. Large solutes also exclude volume and affect processes where biopolymer molecularity and/or shape changes. Here, we develop an analysis to separate and interpret or predict excluded volume and chemical effects of a flexible coil polymer on a process. We report a study of the concentration-dependent effects of the full series from monomeric to polymeric PEG on intramolecular hairpin and intermolecular duplex formation by 12-nucleotide DNA strands. We find that chemical effects of PEG on these processes increase in proportion to the product of the amount of DNA surface exposed on melting and the amount of PEG surface that is accessible to this DNA, and these effects are completely described by two interaction terms that quantify the interactions between this DNA surface and PEG end and interior groups. We find that excluded volume effects, once separated from these chemical effects, are quantitatively described by the analytical theory of Hermans, which predicts the excluded volume between a flexible polymer and a rigid molecule. From this analysis, we show that at constant concentration of PEG monomer, increasing PEG size increases the excluded volume effect but decreases the chemical interaction effect, because in a large PEG coil a smaller fraction of the monomers are accessible to the DNA. Volume exclusion by PEG has a much larger effect on intermolecular duplex formation than on intramolecular hairpin formation. PMID:21742980

  12. Classifying Membrane Proteins in the Proteome by Using Artificial Neural Networks Based on the Preferential Parameters of Amino Acids

    NASA Astrophysics Data System (ADS)

    Bose, Subrata K.; Browne, Antony; Kazemian, Hassan; White, Kenneth

    Membrane proteins (MPs) are large set of biological macromolecules that play a fundamental role in physiology and pathophysiology for survival. From a pharma-economical perspective, though it is the fact that MPs constitute ˜75% of possible targets for novel drugs but MPs are one of the most understudied groups of proteins in biochemical research. This is mainly because of the technical difficulties of obtaining structural information about trans-membrane regions (these are small sequences that crossways the bilayer lipid membrane). It is quite useful to predict the location of transmembrane segments down the sequence, since these are the elementary structural building blocks defining their topology. There have been several attempts over the last 20 years to develop tools for predicting membrane-spanning regions but current tools are far away from achieving a considerable reliability in prediction. This study aims to exploit the knowledge and current understanding in the field of artificial neural networks (ANNs) in particular data representation through the development of a system to identify and predict membrane-spanning regions by analysing primary amino acids sequence. In this paper we present a novel neural network (NNs) architecture and algorithms for predicting membrane spanning regions from primary amino acids sequences by using their preference parameters.

  13. Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics

    PubMed Central

    Chaudhuri, Arunima; Prasanna, Xavier; Agiru, Priyanka; Chakraborty, Hirak; Rydström, Anna; Ho, James C. S.; Svanborg, Catharina; Sengupta, Durba; Chattopadhyay, Amitabha

    2016-01-01

    Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA. PMID:27731329

  14. Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Arunima; Prasanna, Xavier; Agiru, Priyanka; Chakraborty, Hirak; Rydström, Anna; Ho, James C. S.; Svanborg, Catharina; Sengupta, Durba; Chattopadhyay, Amitabha

    2016-10-01

    Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

  15. NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain

    PubMed Central

    Chua, Geok-Lin; Tan, Suet-Mien; Bhattacharjya, Surajit

    2016-01-01

    Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using 15N-1H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane

  16. NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain.

    PubMed

    Chua, Geok-Lin; Tan, Suet-Mien; Bhattacharjya, Surajit

    2016-01-01

    Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using 15N-1H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane

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

    PubMed

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

    2013-10-10

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

  18. Flavonoid-membrane Interactions: A Protective Role of Flavonoids at the Membrane Surface?

    PubMed Central

    Oteiza, Patricia I.; Erlejman, Alejandra G.; Verstraeten, Sandra V.; Keen, Carl L.; Fraga, César G.

    2005-01-01

    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. PMID:15712595

  19. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    NASA Technical Reports Server (NTRS)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  20. Interaction of abscisic acid with phospholipid membranes

    SciTech Connect

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

    1989-04-04

    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. {sup 13}C NMR spin-lattice relaxation times, T{sub 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 {sup 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.

  1. Interaction of ethanol with biological membranes.

    PubMed

    Goldstein, D B; Chin, J H

    1981-05-15

    Ethanol is among the drugs with anesthetic potency determined by lipid solubility, in accord with the Meyer-Overton hypothesis. Thus, it is likely that ethanol acts in a hydrophobic environment. Using electron paramagnetic resonance with 5-doxylstearic acid as spin label, we find that ethanol disorders mouse cell membranes, making the lipid matrix more fluid. We surmise that consequent disruption of the function of integral membrane proteins may be the cause of ethanol's central actions. When mice are treated for 8 days with ethanol, their membranes become tolerant to the disordering effect of ethanol. This tolerance is accompanied by an increased proportion of cholesterol in the membranes.

  2. Outer membrane proteins preferentially load MHC class II peptides: Implications for as a Chlamydia trachomatis T cell vaccine

    PubMed Central

    Karunakaran, Karuna P.; Yu, Hong; Jiang, Xiaozhou; Chan, Queenie; Moon, Kyung-Mee; Foster, Leonard J.; Brunham, Robert C.

    2015-01-01

    CD4 T cell immune responses such as interferon-γ and tumor necrosis factor-α secretion are necessary for Chlamydia immunity. We used an immunoproteomic approach in which Chlamydia trachomatis and Chlamydia muridarum-derived peptides presented by MHC class II molecules on the surface of infected dendritic cells (DCs) were identified by tandem mass spectrometry using bone marrow derived DCs (BMDCs) from mice of different MHC background. We first compared the C. muridarum immunoproteome in C3H mice to that previously identified in C57BL/6 mice. Fourteen MHC class II binding peptides from 11 Chlamydia proteins were identified from C3H infected BMDCs. Two C. muridarum proteins overlapped between C3H and C57B/6 mice and both were polymorphic membrane proteins (Pmps) which presented distinct class II binding peptides. Next we studied DCs from C57BL/6 mice infected with the human strain, C. trachomatis serovar D. Sixty MHC class II binding peptides derived from 27 C. trachomatis proteins were identified. Nine proteins were orthologous T cell antigens between C. trachomatis and C. muridarum and 2 of the nine were Pmps which generated MHC class II binding epitopes at distinct sequences within the proteins. As determined by antigen specific splenocyte responses outer membrane proteins PmpF, -G and -H and the major outer membrane protein (MOMP) were antigenic in mice previously infected with C. muridarum or C. trachomatis. Furthermore a recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with MOMP formulated with a Th1 polarizing adjuvant significantly accelerated (p < 0.001) clearance in the C57BL/6 mice C. trachomatis transcervical infection model. We conclude that Chlamydia outer membrane proteins are important T cell antigens useful in the development of a C. trachomatis subunit vaccine. PMID:25738816

  3. Vortex Interaction on Low Aspect Ratio Membrane Wings

    NASA Astrophysics Data System (ADS)

    Waldman, Rye M.; Breuer, Kenneth S.

    2013-11-01

    Inspired by the flight of bats and by recent interest in Micro Air Vehicles, we present measurements on the steady and unsteady behavior of low aspect ratio membrane wings. We conduct wind tunnel experiments with coupled force, kinematic, and flow field measurements, both on the wing and in the near wake. Membrane wings interact strongly with the vortices shed from the leading- and trailing-edges and the wing tips, and the details of the membrane support play an important role in the fluid-structure interaction. Membranes that are supported at the wing tip exhibit less membrane flutter, more coherent tip vortices, and enhanced lift. The interior wake can exhibit organized spanwise vortex shedding, and shows little influence from the tip vortex. In contrast, membranes with an unsupported wing tip show exaggerated static deformation, significant membrane fluttering and a diffuse, unsteady tip vortex. The unsteady tip vortex modifies the behavior of the interior wake, disrupting the wake coherence.

  4. Membrane interacting regions of Dengue virus NS2A protein.

    PubMed

    Nemésio, Henrique; Villalaín, José

    2014-08-28

    The Dengue virus (DENV) NS2A protein, essential for viral replication, is a poorly characterized membrane protein. NS2A displays both protein/protein and membrane/protein interactions, yet neither its functions in the viral cycle nor its active regions are known with certainty. To highlight the different membrane-active regions of NS2A, we characterized the effects of peptides derived from a peptide library encompassing this protein's full length on different membranes by measuring their membrane leakage induction and modulation of lipid phase behavior. Following this initial screening, one region, peptide dens25, had interesting effects on membranes; therefore, we sought to thoroughly characterize this region's interaction with membranes. This peptide presents an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment. We show that dens25 strongly interacts with membranes that contain a large proportion of lipid molecules with a formal negative charge, and that this effect has a major electrostatic contribution. Considering its membrane modulating capabilities, this region might be involved in membrane rearrangements and thus be important for the viral cycle.

  5. Membrane Interacting Regions of Dengue Virus NS2A Protein

    PubMed Central

    2015-01-01

    The Dengue virus (DENV) NS2A protein, essential for viral replication, is a poorly characterized membrane protein. NS2A displays both protein/protein and membrane/protein interactions, yet neither its functions in the viral cycle nor its active regions are known with certainty. To highlight the different membrane-active regions of NS2A, we characterized the effects of peptides derived from a peptide library encompassing this protein’s full length on different membranes by measuring their membrane leakage induction and modulation of lipid phase behavior. Following this initial screening, one region, peptide dens25, had interesting effects on membranes; therefore, we sought to thoroughly characterize this region’s interaction with membranes. This peptide presents an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment. We show that dens25 strongly interacts with membranes that contain a large proportion of lipid molecules with a formal negative charge, and that this effect has a major electrostatic contribution. Considering its membrane modulating capabilities, this region might be involved in membrane rearrangements and thus be important for the viral cycle. PMID:25119664

  6. Aqua-vanadyl ion interaction with Nafion® membranes

    SciTech Connect

    Vijayakumar, Murugesan; Govind, Niranjan; Li, Bin; Wei, Xiaoliang; Nie, Zimin; Thevuthasan, Suntharampillai; Sprenkle, Vince L.; Wang, Wei

    2015-03-23

    Lack of comprehensive understanding about the interactions between Nafion membrane and battery electrolytes prevents the straightforward tailoring of optimal materials for redox flow battery applications. In this work, we analyzed the interaction between aqua-vanadyl cation and sulfonic sites within the pores of Nafion membranes using combined theoretical and experimental X-ray spectroscopic methods. Molecular level interactions, namely, solvent share and contact pair mechanisms are discussed based on Vanadium and Sulfur K-edge spectroscopic analysis.

  7. Reshaping biological membranes in endocytosis: crossing the configurational space of membrane-protein interactions.

    PubMed

    Simunovic, Mijo; Bassereau, Patricia

    2014-03-01

    Lipid membranes are highly dynamic. Over several decades, physicists and biologists have uncovered a number of ways they can change the shape of membranes or alter their phase behavior. In cells, the intricate action of membrane proteins drives these processes. Considering the highly complex ways proteins interact with biological membranes, molecular mechanisms of membrane remodeling still remain unclear. When studying membrane remodeling phenomena, researchers often observe different results, leading them to disparate conclusions on the physiological course of such processes. Here we discuss how combining research methodologies and various experimental conditions contributes to the understanding of the entire phase space of membrane-protein interactions. Using the example of clathrin-mediated endocytosis we try to distinguish the question 'how can proteins remodel the membrane?' from 'how do proteins remodel the membrane in the cell?' In particular, we consider how altering physical parameters may affect the way membrane is remodeled. Uncovering the full range of physical conditions under which membrane phenomena take place is key in understanding the way cells take advantage of membrane properties in carrying out their vital tasks.

  8. Modeling of interactions between nanoparticles and cell membranes

    NASA Astrophysics Data System (ADS)

    Ban, Young-Min

    Rapid development of nanotechnology and ability to manufacture materials and devices with nanometer feature size leads to exciting innovations in many areas including the medical and electronic fields. However, the possible health and environmental impacts of manufactured nanomaterials are not fully known. Recent experimental reports suggest that some of the manufactured nanomaterials, such as fullerenes and carbon nanotubes, are highly toxic even in small concentrations. The goal of the current work is to understand the mechanisms responsible for the toxicity of nanomaterials. In the current study coarse-grained molecular dynamics simulations are employed to investigate the interactions between NPs and cellular membranes at a molecular level. One of the possible toxicity mechanisms of the nanomaterials is membrane disruption. Possibility of membrane disruption exposed to the manufactured nanomaterials are examined by considering chemical reactions and non-reactive physical interactions as chemical as well as physical mechanisms. Mechanisms of transport of carbon-based nanoparticles (fullerene and its derivative) across a phospholipid bilayer are investigated. The free energy profile is obtained using constrained simulations. It is shown that the considered nanoparticles are hydrophobic and therefore they tend to reside in the interior of the lipid bilayer. In addition, the dynamics of the membrane fluctuations is significantly affected by the nanoparticles at the bilayer-water interface. The hydrophobic interaction between the particles and membrane core induces the strong coupling between the nanoparticle motion and membrane deformation. It is observed that the considered nanoparticles affect several physical properties of the membrane. The nanoparticles embedded into the membrane interior lead to the membrane softening, which becomes more significant with increase in CNT length and concentration. The lateral pressure profile and membrane energy in the membrane

  9. Large-scale identification of yeast integral membrane protein interactions

    PubMed Central

    Miller, John P.; Lo, Russell S.; Ben-Hur, Asa; Desmarais, Cynthia; Stagljar, Igor; Noble, William Stafford; Fields, Stanley

    2005-01-01

    We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions. PMID:16093310

  10. The relevance of membrane models to understand nanoparticles-cell membrane interactions

    NASA Astrophysics Data System (ADS)

    Rascol, Estelle; Devoisselle, Jean-Marie; Chopineau, Joël

    2016-02-01

    Over the past two decades, numerous types of nanoparticles (NPs) have been developed for medical applications; however only a few nanomedicines are actually available on the market. One reason is the lack of understanding and data concerning the NP fate and their behavior upon contact with biological media and cell membranes. Biomimetic membrane models are interesting tools to approach and understand NPs-cell membrane interactions. The use of these models permits one to control physical and chemical parameters and to rapidly compare membrane types and the influence of different media conditions. The interactions between NPs and cell membranes can be qualified and quantified using analytical and modeling methods. In this review, the major studies concerning NPs-cell membrane models and associated methods are described. The advantages and drawbacks for each method are compared for the different models. The key mechanisms of interactions between NPs and cell membranes are revealed using cell membrane models and are interrogated in comparison with the NP behavior in cellulo or in vivo. Investigating the interactions between NPs and cell membrane models is now proposed as an intermediate step between physicochemical characterization of NPs and biological assays.

  11. Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

    NASA Astrophysics Data System (ADS)

    Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

    2009-11-01

    Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

  12. Interaction with membranes of cytochrome c554 from Nitrosomonas europaea.

    PubMed

    McTavish, H; Arciero, D M; Hooper, A B

    1995-12-01

    Two c-cytochromes extrinsically bound to the membranes of Nitrosomonas europaea have been identified. One is the tetraheme cytochrome c554, a protein previously described as soluble and periplasmic. Depending on the concentration of Fe and Cu in the growth medium, from 50 to 100% of the total cellular cytochrome c554 is membrane-associated. The cytochromes c554 found in the soluble or membrane fractions are identical in the spectroscopic, chromatographic, or primary structural properties examined. The interaction of cytochrome c554 with membranes is ionic in nature; it is disrupted by high concentrations of salt. Both membrane-derived and periplasmic forms of cytochrome c554 rebind tightly to membranes which have been washed free of the cytochrome. Cytochrome c554 binds to phospholipid vesicles, suggesting that phospholipids may play a role in the interaction of this cytochrome with the membrane. During the oxidation of NH2OH, the ability of the soluble hydroxylamine oxidoreductase (HAO) to transfer electrons to its natural electron acceptor, cytochrome c554, is substantially impaired when the latter is bound to phospholipid vesicles. The second c-cytochrome associated with membranes in N. europaea is identified as HAO based on its catalytic activity and the presence of a 464-nm ferrous absorption band. A small fraction of HAO is found to be membrane-bound and only in cells grown under low Fe/low Cu. This subpopulation of HAO can be released from the membranes without detergents. PMID:7503559

  13. Misfolding of Amyloidogenic Proteins and Their Interactions with Membranes

    PubMed Central

    Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

    2013-01-01

    In this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. Many of these proteins are implicated in serious illnesses such as Alzheimer’s disease and Parkinson’s disease. Misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. Oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membrane damage. We focus on the structure of these aggregates and their interactions with model membranes. Study of interactions of amlyoidogenic proteins with model and natural membranes has shown the importance of the lipid bilayer in protein misfolding and aggregation and has led to the development of several models for membrane permeabilization by the resulting amyloid aggregates. We discuss several of these models: formation of structured pores by misfolded amyloidogenic proteins, extraction of lipids, interactions with receptors in biological membranes, and membrane destabilization by amyloid aggregates perhaps analogous to that caused by antimicrobial peptides. PMID:24970204

  14. Membrane cholesterol modulates galanin-GalR2 interaction.

    PubMed

    Pang, L; Graziano, M; Wang, S

    1999-09-14

    The neuropeptide galanin mediates a number of diverse physiological and pathophysiological actions via interaction with membrane-bound receptors. The role that membrane cholesterol plays in modulating the interaction between galanin and one of the three cloned galanin receptor subtypes (GalR2) expressed in Chinese hamster ovary (CHO) cells was examined. Reduction of membrane cholesterol by treatment with methyl-beta-cyclodextrin (CD) or by culturing cells in lipoprotein-deficient serum markedly decreased galanin binding to the receptor. Addition of cholesterol back to CD-treated, cholesterol-depleted membranes restored galanin binding to control levels. Hill analysis suggests that the GalR2 binds multiple molecules of cholesterol (n >/= 3) in a positively cooperative manner. This interaction appears to be cholesterol-specific as only cholesterol and a limited number of cholesterol analogues were able to rescue galanin binding. The inability of some of these analogues to rescue the binding activity also suggests that binding of galanin to GalR2 is independent of membrane fluidity as, like cholesterol, cholesterol analogues generally rigidize membranes. In addition, treatment of the membranes with other modulators of membrane fluidity, e.g. ethanol, did not affect galanin binding to the GalR2. In contrast, treatment of membranes, with filipin, a molecule that clusters cholesterol within the membranes, or with cholesterol oxidase resulted in markedly reduced galanin binding. Incubation of membranes with 100 microM GTP-gamma-S did not alter the IC(50) for CD in the prebinding assay treatment suggesting that the effect of cholesterol was independent of G protein interaction. Preincubation of intact cells with CD also drastically impaired the ability of galanin to activate intracellular inositol phosphate accumulation in GalR2-transfected CHO cells. These data detail a new mechanism for the regulation of galanin receptor signaling which may link altered functions of Gal

  15. Modeling Membrane Deformations and Lipid Demixing upon Protein-Membrane Interaction: The BAR Dimer Adsorption

    PubMed Central

    Khelashvili, George; Harries, Daniel; Weinstein, Harel

    2009-01-01

    We use a self-consistent mean-field theory, designed to investigate membrane reshaping and lipid demixing upon interaction with proteins, to explore BAR domains interacting with large patches of lipid membranes of heterogeneous compositions. The computational model includes contributions to the system free energy from electrostatic interactions and elastic energies of the membrane, as well as salt and lipid mixing entropies. The results from our simulation of a single adsorbing Amphiphysin BAR dimer indicate that it is capable of stabilizing a significantly curved membrane. However, we predict that such deformations will occur only for membrane patches that have the inherent propensity for high curvature, reflected in the tendency to create local distortions that closely match the curvature of the BAR dimer itself. Such favorable preconditioning for BAR-membrane interaction may be the result of perturbations such as local lipid demixing induced by the interaction, or of a prior insertion of the BAR domain's amphiphatic N-helix. From our simulations it appears that local segregation of charged lipids under the influence of the BAR dimer cannot produce high enough asymmetry between bilayer leaflets to induce significant bending. In the absence of additional energy contributions that favor membrane asymmetry, the membrane will remain nearly flat upon single BAR dimer adsorption, relative to the undulation expected from thermal fluctuations. Thus, we conclude that the N-helix insertions have a critical mechanistic role in the local perturbation and curving of the membrane, which is then stabilized by the electrostatic interaction with the BAR dimer. We discuss how these results can be used to estimate the tendency of BARs to bend membranes in terms of a spatially nonisotropic spontaneous curvature. PMID:19751667

  16. Fully Quantified Spectral Imaging Reveals in Vivo Membrane Protein Interactions

    PubMed Central

    King, Christopher; Stoneman, Michael; Raicu, Valerica; Hristova, Kalina

    2016-01-01

    Here we introduce the Fully Quantified Spectral Imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling. PMID:26787445

  17. The Role of Cholesterol in Driving IAPP-Membrane Interactions.

    PubMed

    Sciacca, Michele F M; Lolicato, Fabio; Di Mauro, Giacomo; Milardi, Danilo; D'Urso, Luisa; Satriano, Cristina; Ramamoorthy, Ayyalusamy; La Rosa, Carmelo

    2016-07-12

    Our knowledge of the molecular events underlying type 2 diabetes mellitus-a protein conformational disease characterized by the aggregation of islet amyloid polypeptide (IAPP) in pancreatic β cells-is limited. However, amyloid-mediated membrane damage is known to play a key role in IAPP cytotoxicity, and therefore the effects of lipid composition on modulating IAPP-membrane interactions have been the focus of intense research. In particular, membrane cholesterol content varies with aging and consequently with adverse environmental factors such as diet and lifestyle, but its role in the development of the disease is controversial. In this study, we employ a combination of experimental techniques and in silico molecular simulations to shed light on the role of cholesterol in IAPP aggregation and the related membrane disruption. We show that if anionic POPC/POPS vesicles are used as model membranes, cholesterol has a negligible effect on the kinetics of IAPP fibril growth on the surface of the bilayer. In addition, cholesterol inhibits membrane damage by amyloid-induced poration on membranes, but enhances leakage through fiber growth on the membrane surface. Conversely, if 1:2 DOPC/DPPC raft-like model membranes are used, cholesterol accelerates fiber growth. Next, it enhances pore formation and suppresses fiber growth on the membrane surface, leading to leakage. Our results highlight a twofold effect of cholesterol on the amyloidogenicity of IAPP and help explain its debated role in type 2 diabetes mellitus. PMID:27410742

  18. Structural interactions of a voltage sensor toxin with lipid membranes

    PubMed Central

    Mihailescu, Mihaela; Krepkiy, Dmitriy; Milescu, Mirela; Gawrisch, Klaus; Swartz, Kenton J.; White, Stephen

    2014-01-01

    Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor. PMID:25453087

  19. Structural interactions of a voltage sensor toxin with lipid membranes.

    PubMed

    Mihailescu, Mihaela; Krepkiy, Dmitriy; Milescu, Mirela; Gawrisch, Klaus; Swartz, Kenton J; White, Stephen

    2014-12-16

    Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor. PMID:25453087

  20. Influences of acid-base property of membrane on interfacial interactions related with membrane fouling in a membrane bioreactor based on thermodynamic assessment.

    PubMed

    Zhao, Leihong; Qu, Xiaolu; Zhang, Meijia; Lin, Hongjun; Zhou, Xiaoling; Liao, Bao-Qiang; Mei, Rongwu; Hong, Huachang

    2016-08-01

    Failure of membrane hydrophobicity in predicting membrane fouling requires a more reliable indicator. In this study, influences of membrane acid base (AB) property on interfacial interactions in two different interaction scenarios in a submerged membrane bioreactor (MBR) were studied according to thermodynamic approaches. It was found that both the polyvinylidene fluoride (PVDF) membrane and foulant samples in the MBR had relatively high electron donor (γ(-)) component and low electron acceptor (γ(+)) component. For both of interaction scenarios, AB interaction was the major component of the total interaction. The results showed that, the total interaction monotonically decreased with membrane γ(-), while was marginally affected by membrane γ(+), suggesting that γ(-) could act as a reliable indicator for membrane fouling prediction. This study suggested that membrane modification for fouling mitigation should orient to improving membrane surface γ(-) component rather than hydrophilicity. PMID:27155263

  1. Membrane Interaction of the Glycosyltransferase WaaG

    PubMed Central

    Liebau, Jobst; Pettersson, Pontus; Szpryngiel, Scarlett; Mäler, Lena

    2015-01-01

    The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely α-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane. PMID:26244737

  2. Dynamics of a membrane interacting with an active wall.

    PubMed

    Yasuda, Kento; Komura, Shigeyuki; Okamoto, Ryuichi

    2016-05-01

    Active motions of a biological membrane can be induced by nonthermal fluctuations that occur in the outer environment of the membrane. We discuss the dynamics of a membrane interacting hydrodynamically with an active wall that exerts random velocities on the ambient fluid. Solving the hydrodynamic equations of a bound membrane, we first derive a dynamic equation for the membrane fluctuation amplitude in the presence of different types of walls. Membrane two-point correlation functions are calculated for three different cases: (i) a static wall, (ii) an active wall, and (iii) an active wall with an intrinsic time scale. We focus on the mean squared displacement (MSD) of a tagged membrane describing the Brownian motion of a membrane segment. For the static wall case, there are two asymptotic regimes of MSD (∼t^{2/3} and ∼t^{1/3}) when the hydrodynamic decay rate changes monotonically. In the case of an active wall, the MSD grows linearly in time (∼t) in the early stage, which is unusual for a membrane segment. This linear-growth region of the MSD is further extended when the active wall has a finite intrinsic time scale. PMID:27300924

  3. Interaction of gentamicin polycation with model and cell membranes.

    PubMed

    Kovács, Eugenia; Savopol, Tudor; Iordache, Maria-Minodora; Săplăcan, Lavinia; Sobaru, Iuliana; Istrate, Claudia; Mingeot-Leclercq, Marie-Paule; Moisescu, Mihaela-Georgeta

    2012-10-01

    The interaction of positively-charged antibiotic gentamicin with cell membranes was studied to determine if any changes in membrane organization were induced by the drug. Opossum kidney epithelia (OK) cells were used as models of eukaryotic cells. Two methods were used: laurdan fluorescence spectroscopy and fluorescence anisotropy recordings on 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) labeled cell suspensions. Both methods showed an altered membrane hydration and fluidity of gentamicin treated cells. Liposomes prepared from dimyristoyl-phosphatidylcholine (DMPC) mixed with cardiolipin, which mimics the heterogeneous charge composition of the natural cell membrane, were used to determine the effect of gentamicin on artificial bilayers. The membrane lipid packing as revealed by generalized polarization (GP) and fluorescence anizotropy variation with increasing temperature was studied. It was found that the generalized polarization of liposomal membranes containing a negatively charged lipid (cardiolipin) is higher in the presence of gentamicin; in the membrane of living cell (OK), gentamicin induces, on the contrary, a decrease of general polarization. Considering the role of membrane organization in the function of transmembrane channels and receptors, our findings suggest hypotheses that may explain the permeation of gentamicin through the living cell membrane by using these channels.

  4. [Interaction of melittin with ion channels of excitable membranes].

    PubMed

    Zherelova, O M; Kabanova, N V; Kazachenko, V N; Chaĭlakhian, L M

    2007-01-01

    The effect of the neurotoxin melittin on the activation of ion channels of excitable membrane, the plasmalemma of Characeae algae cells, isolated membrane patches of neurons of mollusc L. stagnalis and Vero cells was studied by the method of intracellular perfusion and the patch-clamp technique in inside-out configuration. It was shown that melittin disturbs the conductivity of plasmalemma and modifieds Ca(2+)-channels of plant membrane. The leakage current that appears by the action of melittin can be restored by substituting calmodulin for melittin. Melittin modifies K(+)-channels of animal cell membrane by disrupting the phospholipid matrix and forms conductive structures in the membrane by interacting with channel proteins, which is evidenced by the appearance of additional ion channels.

  5. [Interaction of melittin with ion channels of excitable membranes].

    PubMed

    Zherelova, O M; Kabanova, N V; Kazachenko, V N; Chaĭlakhian, L M

    2007-01-01

    The effect of the neurotoxin melittin on the activation of ion channels of excitable membrane, the plasmalemma of Characeae algae cells, isolated membrane patches of neurons of mollusc L. stagnalis and Vero cells was studied by the method of intracellular perfusion and the patch-clamp technique in inside-out configuration. It was shown that melittin disturbs the conductivity of plasmalemma and modifieds Ca(2+)-channels of plant membrane. The leakage current that appears by the action of melittin can be restored by substituting calmodulin for melittin. Melittin modifies K(+)-channels of animal cell membrane by disrupting the phospholipid matrix and forms conductive structures in the membrane by interacting with channel proteins, which is evidenced by the appearance of additional ion channels. PMID:17477057

  6. The anticancer drug cisplatin interacts with the human erythrocyte membrane.

    PubMed

    Suwalsky, M; Hernández, P; Villena, F; Sotomayor, C P

    2000-01-01

    Drugs which exert their effects by interacting with DNA cause structural and functional membrane alterations which may be essential for growth inhibition by these agents. This paper describes the interaction of cisplatin with the human erythrocyte membrane and models constituted by bilayers of dimyristoylphosphatidylethanolamine (DMPE) and diacylphosphatidylserine (DAPS), representative of phospholipid classes located in the inner monolayer of the erythrocyte membrane, and of dimyristoylphosphatidylcholine (DMPC), a class present in its outer monolayer. Cisplatin ability to perturb DMPE, DAPS and DMPC bilayer structures was determined by X-ray diffraction and fluorescence spectroscopy. Electron microscopy disclosed that human erythrocytes incubated with 35 microM cisplatin, which is its therapeutical concentration in serum, developed cup-shaped forms (stomatocytes). According to the bilayer couple hypothesis, this means that the drug is inserted into the inner monolayer of the erythrocyte membrane, a conclusion supported by the studies on model systems. PMID:10928560

  7. Probing the interaction of amphiphilic triblockcopolymers with a biomimetic membrane.

    SciTech Connect

    Firestone, M. A.; Seifert, S.

    2002-02-04

    In the last several years, there has been growing interest in the use of synthetic surfactants to augment cellular repair. Amphiphilic triblock copolymers such as PEO-PPO-PEO have been demonstrated to aid in the repair of a variety of cells. In spite of the reported success of these compounds in clinical trials, the mechanism of their interaction with cell membranes remains poorly understood. In this work, they describe their efforts to examine the effect of the mode of incorporation of triblock polyalkyleneoxide copolymers on membrane structure and stability. For this work, they have employed a model biomembrane whose structure and physical properties have been previously determined. Several modes of polymer incorporation are examined: introduction via a membrane spanning triblock copolymer, grafting onto a phospholipid headgroup, or introduction via a partially inserted triblock copolymer. The polymer-membrane interactions are probed by small angle X-ray scattering and thermal analysis.

  8. Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

    PubMed Central

    Gauliard, Emilie; Ouellette, Scot P.; Rueden, Kelsey J.; Ladant, Daniel

    2015-01-01

    Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins). Incs represent 7–10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH) method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF, and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia. PMID:25717440

  9. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  10. Interactions between model bacterial membranes and synthetic antimicrobials.

    NASA Astrophysics Data System (ADS)

    Yang, Lihua; Mishra, Abhijit; Som, Abhigyan; Tew, Gregory N.; Wong, Gerard C. L.

    2006-03-01

    Antimicrobial peptides comprise a key component of innate immunity for a wide range of multicellular organisms. It has been shown that natural antimicrobial peptides and their analogs can permeate bacterial membranes selectively. There are a number of proposed models for this action, but the detailed molecular mechanism of the induced membrane permeation remains unclear. We investigate interactions between model bacterial membranes and a prototypical family of phenylene ethynylene-based antimicrobials with controllable hydrophilic and hydrophobic volume fractions, controllable charge placement. Preliminary results from synchrotron small angle x-ray scattering (SAXS) results will be presented.

  11. Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern

    PubMed Central

    Lefrançois, Philippe; Rockmill, Beth; Xie, Pingxing; Roeder, G. Shirleen; Snyder, Michael

    2016-01-01

    During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required. The nature and mechanism of coupling have yet to be elucidated. Here we present the first pairwise analysis of centromere coupling in an effort to uncover underlying rules that may exist within these non-homologous interactions. We designed a novel chromosome conformation capture (3C)-based assay to detect all possible interactions between non-homologous yeast centromeres during early meiosis. Using this variant of 3C-qPCR, we found a size-dependent interaction pattern, in which chromosomes assort preferentially with chromosomes of similar sizes, in haploid and diploid spo11 cells, but not in a coupling-defective mutant (spo11 zip1 haploid and diploid yeast). This pattern is also observed in wild-type diploids early in meiosis but disappears as meiosis progresses and homologous chromosomes pair. We found no evidence to support the notion that ancestral centromere homology plays a role in pattern establishment in S. cerevisiae post-genome duplication. Moreover, we found a role for the meiotic bouquet in establishing the size dependence of centromere coupling, as abolishing bouquet (using the bouquet-defective spo11 ndj1 mutant) reduces it. Coupling in spo11 ndj1 rather follows telomere clustering preferences. We propose that a chromosome size preference for centromere coupling helps establish efficient homolog recognition. PMID:27768699

  12. Interaction of chiral rafts in self-assembled colloidal membranes

    NASA Astrophysics Data System (ADS)

    Xie, Sheng; Hagan, Michael F.; Pelcovits, Robert A.

    2016-03-01

    Colloidal membranes are monolayer assemblies of rodlike particles that capture the long-wavelength properties of lipid bilayer membranes on the colloidal scale. Recent experiments on colloidal membranes formed by chiral rodlike viruses showed that introducing a second species of virus with different length and opposite chirality leads to the formation of rafts—micron-sized domains of one virus species floating in a background of the other viruses [Sharma et al., Nature (London) 513, 77 (2014), 10.1038/nature13694]. In this article we study the interaction of such rafts using liquid crystal elasticity theory. By numerically minimizing the director elastic free energy, we predict the tilt angle profile for both a single raft and two rafts in a background membrane, and the interaction between two rafts as a function of their separation. We find that the chiral penetration depth in the background membrane sets the scale for the range of the interaction. We compare our results with the experimental data and find good agreement for the strength and range of the interaction. Unlike the experiments, however, we do not observe a complete collapse of the data when rescaled by the tilt angle at the raft edge.

  13. Theoretical study of interactions between striated cylindrical particles and membrane

    NASA Astrophysics Data System (ADS)

    Wang, Jing-Jing; Jia-Wei, Feng; Ren, Chun-Lai

    2015-08-01

    The interaction of nanoparticles with cell membranes is of great importance because of their potential biomedical applications. In this paper, we investigate the adhesion of stripe-patterned cylinders to a fluid membrane with a full consideration of the Helfrich free energy. Three situations are considered: one striated cylindrical particle, two pure cylindrical particles, and two Janus cylindrical particles. It is found that, with the adhesion of a single sparse striated cylinder, there are a variety of steady-states with energy barriers and the stable state is determined by the pattern of the cylinder. However, when the particle is densely striped, it has no effect on the stable state. By comparing the wrapping degree of two cylindrical particles with that of a single cylindrical particle, we find that two pure cylindrical particles can promote or suppress their interaction with the membrane under different situations. However, two Janus cylindrical particles can only inhibit their interaction with the membrane. Besides, this interaction is related to a first-order transition which is a shallow-to-deep wrapping transition for two pure cylinders while it is a shallow-to-half wrapping transition for two Janus cylinders. Furthermore, the position where the transition happens as a function of adhesion energy is given for fixed membrane tension and the precondition of the transition is presented. Project supported by the National Natural Science Foundation of China (Grant Nos. 91027040 and 21274062).

  14. Interaction of mammalian Hsp22 with lipid membranes

    PubMed Central

    Chowdary, Tirumala Kumar; Raman, Bakthisaran; Ramakrishna, Tangirala; Rao, Ch. Mohan

    2006-01-01

    Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro. Intrinsic tryptophan fluorescence is quenched in the presence of lipid vesicles derived from either bovine brain lipid extract or purified lipids. Time-resolved fluorescence studies show a decrease in the lifetimes of the tryptophan residues. Both of these results indicate burial of some tryptophan residues of Hsp22 upon interaction with lipid vesicles. Membrane interactions also lead to increase in fluorescence polarization of Hsp22. Gel-filtration chromatography shows that Hsp22 binds stably with lipid vesicles; the extent of binding depends on the nature of the lipid. Hsp22 binds more strongly to vesicles made of lipids containing a phosphatidic acid, phosphatidylinositol or phosphatidylserine headgroup (known to be present in the inner leaflet of plasma membrane) compared with lipid vesicles made of a phosphatidylcholine head-group alone. Far-UV CD spectra reveal conformational changes upon binding to the lipid vesicles or in membrane-mimetic solvent, trifluoroethanol. Thus our fluorescence, CD and gel-filtration studies show that Hsp22 interacts with membrane and this interaction leads to stable binding and conformational changes. The present study therefore clearly demonstrates that Hsp22 exhibits potential membrane interaction that may play an important role in its cellular functions. PMID:17020537

  15. Interactions of Lipidic Cubic Phase Nanoparticles with Lipid Membranes.

    PubMed

    Jabłonowska, Elżbieta; Nazaruk, Ewa; Matyszewska, Dorota; Speziale, Chiara; Mezzenga, Raffaele; Landau, Ehud M; Bilewicz, Renata

    2016-09-20

    The interactions of liquid-crystalline monoolein (GMO) cubic phase nanoparticles with various model lipid membranes spread at the air-solution interface by the Langmuir technique were investigated. Cubosomes have attracted attention as potential biocompatible drug delivery systems, and thus understanding their mode of interaction with membranes is of special interest. Cubosomes spreading at the air-water interface as well as interactions with a monolayer of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) compressed to different surface pressures were studied by monitoring surface pressure-time dependencies at constant area. Progressive incorporation of the nanoparticles was shown to lead to mixed monolayer formation. The concentration of cubosomes influenced the mechanism of incorporation, as well as the fluidity and permeability of the resulting lipid membranes. Brewster angle microscopy images reflected the dependence of the monolayer structure on the cubosomes presence in the subphase. A parameter Csat was introduced to indicate the point of saturation of the lipid membrane with the cubosomal material. This parameter was found to depend on the surface pressure showing that the cubosomes disintegrate in prolonged contact with the membrane, filling available voids in the lipid membrane. At highest surface pressures when the layer is most compact, the penetration of cubosomal material is not possible and only some exchange with the membrane lipid becomes the route of including GMO into the layer. Finally, comparative studies of the interactions between lipids with various headgroup charges with cubosomes suggest that at high surface pressure an exchange of lipid component between the monolayer and the cubosome in its intact form may occur. PMID:27550742

  16. Interactions of Lipidic Cubic Phase Nanoparticles with Lipid Membranes.

    PubMed

    Jabłonowska, Elżbieta; Nazaruk, Ewa; Matyszewska, Dorota; Speziale, Chiara; Mezzenga, Raffaele; Landau, Ehud M; Bilewicz, Renata

    2016-09-20

    The interactions of liquid-crystalline monoolein (GMO) cubic phase nanoparticles with various model lipid membranes spread at the air-solution interface by the Langmuir technique were investigated. Cubosomes have attracted attention as potential biocompatible drug delivery systems, and thus understanding their mode of interaction with membranes is of special interest. Cubosomes spreading at the air-water interface as well as interactions with a monolayer of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) compressed to different surface pressures were studied by monitoring surface pressure-time dependencies at constant area. Progressive incorporation of the nanoparticles was shown to lead to mixed monolayer formation. The concentration of cubosomes influenced the mechanism of incorporation, as well as the fluidity and permeability of the resulting lipid membranes. Brewster angle microscopy images reflected the dependence of the monolayer structure on the cubosomes presence in the subphase. A parameter Csat was introduced to indicate the point of saturation of the lipid membrane with the cubosomal material. This parameter was found to depend on the surface pressure showing that the cubosomes disintegrate in prolonged contact with the membrane, filling available voids in the lipid membrane. At highest surface pressures when the layer is most compact, the penetration of cubosomal material is not possible and only some exchange with the membrane lipid becomes the route of including GMO into the layer. Finally, comparative studies of the interactions between lipids with various headgroup charges with cubosomes suggest that at high surface pressure an exchange of lipid component between the monolayer and the cubosome in its intact form may occur.

  17. A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes*

    PubMed Central

    Chu, Nam K.; Shabbir, Waheed; Bove-Fenderson, Erin; Araman, Can; Lemmens-Gruber, Rosa; Harris, David A.; Becker, Christian F. W.

    2014-01-01

    Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrPC into pathogenic PrPSc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions. PMID:25217642

  18. A C-terminal membrane anchor affects the interactions of prion proteins with lipid membranes.

    PubMed

    Chu, Nam K; Shabbir, Waheed; Bove-Fenderson, Erin; Araman, Can; Lemmens-Gruber, Rosa; Harris, David A; Becker, Christian F W

    2014-10-24

    Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

  19. The cellular membrane as a mediator for small molecule interaction with membrane proteins.

    PubMed

    Mayne, Christopher G; Arcario, Mark J; Mahinthichaichan, Paween; Baylon, Javier L; Vermaas, Josh V; Navidpour, Latifeh; Wen, Po-Chao; Thangapandian, Sundarapandian; Tajkhorshid, Emad

    2016-10-01

    The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:27163493

  20. A Phase 1 Study of a Vaccine Targeting Preferentially Expressed Antigen in Melanoma and Prostate-specific Membrane Antigen in Patients With Advanced Solid Tumors

    PubMed Central

    Weber, Jeffrey S.; Vogelzang, Nicholas J.; Ernstoff, Marc S.; Goodman, Oscar B.; Cranmer, Lee D.; Marshall, John L.; Miles, Sabrina; Rosario, Dar; Diamond, David C.; Qiu, Zhiyong; Obrocea, Mihail; Bot, Adrian

    2013-01-01

    Summary Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications. PMID:21760528

  1. Probing DNA-lipid membrane interactions with a lipopeptide nanopore.

    PubMed

    Bessonov, Andrey; Takemoto, Jon Y; Simmel, Friedrich C

    2012-04-24

    Association of DNA molecules with lipid bilayer membranes is of considerable interest for a large variety of applications in biotechnology. Here we introduce syringomycin E (SRE), a small pore-forming lipopeptide produced by the bacterium Pseudomonas syringae, as a facile sensor for the detection of DNA interactions with lipid membranes. SRE forms highly reproducible pores in cellular and artificial membranes. The pore structure involves bilayer lipids, which have a pronounced influence on open channel conductance and gating. SRE channels act as ionic diodes that serve as current rectifiers sensitive to the charge of the bilayer. We employ this intrinsic property to electronically monitor the association of DNA molecules with the membrane in a variety of different settings. We show that SRE can be used for quantitatively probing electrostatic interactions of DNA and DNA-cholesterol conjugates with a lipid membrane. Furthermore, we demonstrate that SRE channels allow monitoring of hybridization reactions between lipid-anchored probe strands and complementary strands in solution. In the presence of double-stranded DNA, SRE channels display a particularly high degree of rectification. Finally, the formation of multilayered structures assembled from poly-(L)-lysine and DNA oligonucleotides on the membrane was precisely monitored with SRE. PMID:22424398

  2. Direct simulation of amphiphilic nanoparticle mediated membrane interactions

    NASA Astrophysics Data System (ADS)

    Tahir, Mukarram; Alexander-Katz, Alfredo

    Membrane fusion is a critical step in the transport of biological cargo through membrane-bound compartments like vesicles. Membrane proteins that alleviate energy barriers for initial stalk formation and eventual rupture of the hemifusion intermediate during fusion generally assist this process. Gold nanoparticles functionalized with a combination of hydrophobic and hydrophilic alkanethiol ligands have recently been shown to induce membrane re-arrangements that are similar to those associated with these fusion proteins. In this work, we utilize molecular dynamics simulation to systematically design nanoparticles that exhibit targeted interactions with membranes. We introduce a method for rapidly parameterizing nanoparticle topology for the MARTINI biomolecular force field to permit long timescale simulation of their interactions with lipid bilayers. We leverage this model to investigate how ligand chemistry governs the nanoparticle's insertion efficacy and the perturbations it generates in the membrane environment. We further demonstrate through unbiased simulations that these nanoparticles can direct the fusion of lipid assemblies such as micelles and vesicles in a manner that mimics the function of biological fusion peptides and SNARE proteins.

  3. Electrostatic interactions of asymmetrically charged membranes

    NASA Astrophysics Data System (ADS)

    Ben-Yaakov, Dan; Burak, Yoram; Andelman, David; Safran, S. A.

    2007-08-01

    We predict the nature (attractive or repulsive) and range (exponentially screened or long-range power law) of the electrostatic interactions of oppositely charged, planar plates as a function of the salt concentration and surface charge densities (whose absolute magnitudes are not necessarily equal). An analytical expression for the crossover between attractive and repulsive pressure is obtained as a function of the salt concentration. This condition reduces to the high-salt limit of Parsegian and Gingell where the interaction is exponentially screened and to the zero salt limit of Lau and Pincus in which the important length scales are the inter-plate separation and the Gouy-Chapman length. In the regime of low salt and high surface charges we predict —for any ratio of the charges on the surfaces— that the attractive pressure is long-ranged as a function of the spacing. The attractive pressure is related to the decrease in counter-ion concentration as the inter-plate distance is decreased. Our theory predicts several scaling regimes with different scaling expressions for the pressure as a function of salinity and surface charge densities. The pressure predictions can be related to surface force experiments of oppositely charged surfaces that are prepared by coating one of the mica surfaces with an oppositely charged polyelectrolyte.

  4. Interaction measurement of particles bound to a lipid membrane

    NASA Astrophysics Data System (ADS)

    Sarfati, Raphael; Dufresne, Eric

    2015-03-01

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

  5. Effects of surface charge on interfacial interactions related to membrane fouling in a submerged membrane bioreactor based on thermodynamic analysis.

    PubMed

    Cai, Huihui; Fan, Hao; Zhao, Leihong; Hong, Huachang; Shen, Liguo; He, Yiming; Lin, Hongjun; Chen, Jianrong

    2016-03-01

    Effects of both membrane and sludge foulant surface zeta potentials on interfacial interactions between membrane and sludge foulant in different interaction scenarios were systematically investigated based on thermodynamic methods. Under conditions in this study, it was found that zeta potential had marginal effects on total interfacial interaction between two infinite planar surfaces, and the total interfacial interaction between foulant particles and membrane would be more repulsive with increase of absolute value of zeta potential. Adhesion of foulant particles on membrane surface should overcome an energy barrier. There exists a critical zeta potential below which energy barrier would disappear. Results also showed that rough surface membrane corresponded to significantly low strength of interfacial interactions. This study not only provided a series of methods to quantitatively assess the interfacial interactions between membrane and sludge foulants, but also reconciled the contradictory conclusions regarding effects of zeta potential in literature, giving important implications for membrane fouling mitigation.

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

    PubMed

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

    2014-02-25

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

  7. Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains.

    PubMed

    Zerbes, Ralf M; Bohnert, Maria; Stroud, David A; von der Malsburg, Karina; Kram, Anita; Oeljeklaus, Silke; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils; Veenhuis, Marten; van der Klei, Ida J; Pfanner, Nikolaus; van der Laan, Martin

    2012-09-14

    The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.

  8. Galactocerebroside-phospholipid interactions in bilayer membranes.

    PubMed

    Ruocco, M J; Shipley, G G; Oldfield, E

    1983-07-01

    Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.

  9. ENaC-membrane interactions: regulation of channel activity by membrane order.

    PubMed

    Awayda, Mouhamed S; Shao, Weijian; Guo, Fengli; Zeidel, Mark; Hill, Warren G

    2004-06-01

    Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459-6463). As these changes of temperature affect membrane lipid order and lipid-protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order-dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2 degrees C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of

  10. Electrostatic interaction of neutral semi-permeable membranes

    NASA Astrophysics Data System (ADS)

    Vinogradova, Olga I.; Bocquet, Lyderic; Bogdanov, Artem N.; Tsekov, Roumen; Lobaskin, Vladimir

    2012-01-01

    We consider an osmotic equilibrium between bulk solutions of polyelectrolyte bounded by semi-permeable membranes and separated by a thin film of salt-free liquid. Although the membranes are neutral, the counter-ions of the polyelectrolyte molecules permeate into the gap and lead to a steric charge separation. This gives rise to a distance-dependent membrane potential, which translates into a repulsive electrostatic disjoining pressure. From the solution of the nonlinear Poisson-Boltzmann equation, we obtain the distribution of the potential and of ions. We then derive an explicit formula for the pressure exerted on the membranes and show that it deviates from the classical van't Hoff expression for the osmotic pressure. This difference is interpreted in terms of a repulsive electrostatic disjoining pressure originating from the overlap of counterion clouds inside the gap. We also develop a simplified theory based on a linearized Poisson-Boltzmann approach. A comparison with simulation of a primitive model for the electrolyte is provided and does confirm the validity of the theoretical predictions. Beyond the fundamental result that the neutral surfaces can repel, this mechanism not only helps to control the adhesion and long-range interactions of living cells, bacteria, and vesicles, but also allows us to argue that electrostatic interactions should play enormous role in determining behavior and functions of systems bounded by semi-permeable membranes.

  11. Electrostatic interaction of neutral semi-permeable membranes.

    PubMed

    Vinogradova, Olga I; Bocquet, Lyderic; Bogdanov, Artem N; Tsekov, Roumen; Lobaskin, Vladimir

    2012-01-21

    We consider an osmotic equilibrium between bulk solutions of polyelectrolyte bounded by semi-permeable membranes and separated by a thin film of salt-free liquid. Although the membranes are neutral, the counter-ions of the polyelectrolyte molecules permeate into the gap and lead to a steric charge separation. This gives rise to a distance-dependent membrane potential, which translates into a repulsive electrostatic disjoining pressure. From the solution of the nonlinear Poisson-Boltzmann equation, we obtain the distribution of the potential and of ions. We then derive an explicit formula for the pressure exerted on the membranes and show that it deviates from the classical van't Hoff expression for the osmotic pressure. This difference is interpreted in terms of a repulsive electrostatic disjoining pressure originating from the overlap of counterion clouds inside the gap. We also develop a simplified theory based on a linearized Poisson-Boltzmann approach. A comparison with simulation of a primitive model for the electrolyte is provided and does confirm the validity of the theoretical predictions. Beyond the fundamental result that the neutral surfaces can repel, this mechanism not only helps to control the adhesion and long-range interactions of living cells, bacteria, and vesicles, but also allows us to argue that electrostatic interactions should play enormous role in determining behavior and functions of systems bounded by semi-permeable membranes.

  12. Nucleic acid-lipid membrane interactions studied by DSC

    PubMed Central

    Giatrellis, Sarantis; Nounesis, George

    2011-01-01

    The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA—lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid—membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies. PMID:21430956

  13. The anticancer drug adriamycin interacts with the human erythrocyte membrane.

    PubMed

    Suwalsky, M; Hernández, P; Villena, F; Aguilar, F; Sotomayor, C P

    1999-01-01

    Adriamycin is an aminoglycosidic anthracycline antibiotic widely used in the treatment of cancer. Increasing reports point to the involvement of cell membranes in its mechanism of action. The interaction of adriamycin with human erythrocytes was investigated in order to determine the membrane binding sites and the resultant structural perturbation. Electron microscopy revealed that red cells incubated with the therapeutical concentration of the drug in human plasma changed their discoid shape to both stomatocytes and echinocytes. According to the bilayer couple hypothesis, this means that adriamycin was incorporated into either the inner or outer leaflets of the erythrocyte membrane. To explain this unusual result, the drug was incubated with molecular models. One of them consisted of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) multilayers, representative of phospholipid classes located in the outer and inner leaflets of the erythrocyte membrane, respectively. X-ray diffraction showed that adriamycin interaction perturbed the polar head and acyl chain regions of both lipids. Fluorescence spectroscopy on another model, consisting of DMPC large unilamellar vesicles (LUV), confirmed the X-ray results in that adriamycin fluidized its hydrophobic moiety. It is concluded that adriamycin incorporates into both erythrocyte leaflets affecting its membrane structure. PMID:10349743

  14. Peptide-membrane Interactions by Spin-labeling EPR

    PubMed Central

    Smirnova, Tatyana I.; Smirnov, Alex I.

    2016-01-01

    Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described. PMID:26477253

  15. Peptide-Membrane Interactions by Spin-Labeling EPR.

    PubMed

    Smirnova, Tatyana I; Smirnov, Alex I

    2015-01-01

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described.

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

    PubMed

    Giatrellis, Sarantis; Nounesis, George

    2011-01-01

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

  17. Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions

    PubMed Central

    Chavent, Matthieu; Seiradake, Elena; Jones, E. Yvonne; Sansom, Mark S.P.

    2016-01-01

    Summary Ephs are transmembrane receptors that mediate cell-cell signaling. The N-terminal ectodomain binds ligands and enables receptor clustering, which activates the intracellular kinase. Relatively little is known about the function of the membrane-proximal fibronectin domain 2 (FN2) of the ectodomain. Multiscale molecular dynamics simulations reveal that FN2 interacts with lipid bilayers via a site comprising K441, R443, R465, Q462, S464, S491, W467, F490, and P459–461. FN2 preferentially binds anionic lipids, a preference that is reduced in the mutant K441E + R443E. We confirm these results by measuring the binding of wild-type and mutant FN2 domains to lipid vesicles. In simulations of the complete EphA2 ectodomain plus the transmembrane region, we show that FN2 anchors the otherwise flexible ectodomain at the surface of the bilayer. Altogether, our data suggest that FN2 serves a dual function of interacting with anionic lipids and constraining the structure of the EphA2 ectodomain to adopt membrane-proximal configurations. PMID:26724997

  18. Revisiting the membrane interaction mechanism of a membrane-damaging β-barrel pore-forming toxin Vibrio cholerae cytolysin.

    PubMed

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2015-09-01

    Vibrio cholerae cytolysin (VCC) permeabilizes target cell membranes by forming transmembrane oligomeric β-barrel pores. VCC has been shown to associate with the target membranes via amphipathicity-driven spontaneous partitioning into the membrane environment. More specific interaction(s) of VCC with the membrane components have also been documented. In particular, specific binding of VCC with the membrane lipid components is believed to play a crucial role in determining the efficacy of the pore-formation process. However, the structural basis and the functional implications of the VCC interaction with the membrane lipids remain unclear. Here we show that the distinct loop sequences within the membrane-proximal region of VCC play critical roles to determine the functional interactions of the toxin with the membrane lipids. Alterations of the loop sequences via structure-guided mutagenesis allow amphipathicity-driven partitioning of VCC to the membrane lipid bilayer. Alterations of the loop sequences, however, block specific interactions of VCC with the membrane lipids and abort the oligomerization, membrane insertion, pore-formation and cytotoxic activity of the toxin. Present study identifies the structural signatures in VCC implicated for its functional interactions with the membrane lipid components, a process that presumably acts to drive the subsequent steps of the oligomeric β-barrel pore-formation and cytotoxic responses.

  19. Coarse-Grained Models for Protein-Cell Membrane Interactions

    PubMed Central

    Bradley, Ryan; Radhakrishnan, Ravi

    2015-01-01

    The physiological properties of biological soft matter are the product of collective interactions, which span many time and length scales. Recent computational modeling efforts have helped illuminate experiments that characterize the ways in which proteins modulate membrane physics. Linking these models across time and length scales in a multiscale model explains how atomistic information propagates to larger scales. This paper reviews continuum modeling and coarse-grained molecular dynamics methods, which connect atomistic simulations and single-molecule experiments with the observed microscopic or mesoscale properties of soft-matter systems essential to our understanding of cells, particularly those involved in sculpting and remodeling cell membranes. PMID:26613047

  20. Cell membrane interaction of Bacillus thuringiensis subsp. israelensis cytolytic toxins.

    PubMed

    Gill, S S; Singh, G J; Hornung, J M

    1987-05-01

    Two toxic polypeptides of 24 and 25 kilodaltons (kDa) were purified from parasporal proteinaceous crystals of Bacillus thuringiensis subsp. israelensis. Both of these polypeptides, which are antigenically similar and have identical N terminals, lysed human erythrocytes and cultured mosquito cells. Although the 24-kDa peptide was more toxic than the 25-kDa peptide, both were less toxic than the crude alkali-solubilized crystal toxin. However, a 1:1 mixture of these 24- and 25-kDa proteins was more toxic than either of these polypeptides individually, indicating a possible interaction between these proteins at the cell membrane. Both the 24- and the 25-kDa proteins were inactivated by aqueous suspensions of dioleolylphosphatidylcholine, indicating the involvement of phospholipids in the cytotoxic action of these toxins. Thus the role of cell membrane phospholipids in mediating the toxin action was studied by using phospholipases as probes. Treatment of erythrocytes with high levels of phospholipase D increased their susceptibility to the toxin; however, phospholipase A2-treated erythrocytes were less susceptible to the toxin. These erythrocytes also bound less 125I-labeled 25-kDa toxin. These results support the role of fatty acyl residues at the syn-2 position of membrane phospholipids in toxin action. The cytolytic toxin of B. thuringiensis subsp. israelensis is thought to damage cell membranes in a detergentlike manner. However, there was a difference between the cytolytic action of this toxin and that of a nonionic detergent such as Triton X-100 because phospholipase A2-treated erythrocytes were more susceptible to Triton X-100, whereas such erythrocytes were less sensitive to the toxin. Thus, the cytolytic toxin apparently did not act as a nonspecific detergent, but rather interacted with phospholipid receptors on the cell membrane. Such an interaction of the toxin with phospholipid receptors probably results in the increased cell permeability, thereby causing

  1. Lipopolysaccharide-induced hemolysis: Evidence for direct membrane interactions

    PubMed Central

    Brauckmann, Stephan; Effenberger-Neidnicht, Katharina; de Groot, Herbert; Nagel, Michael; Mayer, Christian; Peters, Jürgen; Hartmann, Matthias

    2016-01-01

    While hemolysis in patients with sepsis is associated with increased mortality its mechanisms are unknown and Toll-like receptor (TLR)-4 mediated effects, complement-mediated hemolysis, or direct cell membrane effects are all conceivable mechanisms. In this study, we tested the hypotheses that toxic lipopolysaccharide (LPS) as well as non-toxic RS-LPS evokes hemolysis (1) by direct membrane effects, and (2) independent of the complement system and TLR-4 activation. We found, that incubation with LPS resulted in a marked time and concentration dependent increase of free hemoglobin concentration and LDH activity in whole blood and washed red cells. Red cell integrity was diminished as shown by decreased osmotic resistance, formation of schistocytes and rolls, and a decrease in red cell membrane stiffness. Non-toxic RS-LPS inhibited the LPS-evoked increase in TNF-α concentration demonstrating its TLR-4 antagonism, but augmented LPS-induced increase in supernatant hemoglobin concentration and membrane disturbances. Removal of plasma components in washed red cell assays failed to attenuate hemolysis. In summary, this study demonstrates direct physicochemical interactions of LPS with red cell membranes resulting in hemolysis under in vitro conditions. It might thus be hypothesized, that not all effects of LPS are mediated by TLR and may explain LPS toxicity in cells missing TLR. PMID:27759044

  2. Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

    PubMed Central

    Khadka, Nawal K.; Cheng, Xiaolin; Ho, Chian Sing; Katsaras, John; Pan, Jianjun

    2015-01-01

    Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus KA is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM. PMID:25992727

  3. Interaction of Sindbis virus with liposomal model membranes.

    PubMed Central

    Mooney, J J; Dalrymple, J M; Alving, C R; Russell, P K

    1975-01-01

    Radiolabeled Sindbis virus was found to bind to protein-free lipid model membranes (liposomes) derived from extracts of sheep erythrocytes. The virus interaction was dependent on initial pH, and the range of pH dependence (pH 6.0 to 6.8) was the same as the observed with virus-dependent hemagglutination. After the initial interaction, pH changes no longer influenced the virus binding to liposomes. Virus bound to liposomes prepared from a mixture of erythrocyte phospholipids, but the binding was greatly diminished when either cholesterol or phosphatidylethanolamine was omitted from the liposomal lipid mixture. It was concluded that phospholipids and cholesterol, in a bilayer configuration, may be sufficient for specific virus binding in the absence of membrane protein. PMID:234538

  4. Membrane and inhibitor interactions of intracellular phospholipases A2.

    PubMed

    Mouchlis, Varnavas D; Dennis, Edward A

    2016-05-01

    Studying phospholipases A2 (PLA2s) is a challenging task since they act on membrane-like aggregated substrates and not on monomeric phospholipids. Multidisciplinary approaches that include hydrogen/deuterium exchange mass spectrometry (DXMS) and computational techniques have been employed with great success in order to address important questions about the mode of interactions of PLA2 enzymes with membranes, phospholipid substrates and inhibitors. Understanding the interactions of PLA2s is crucial since these enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid (AA) and other polyunsaturated fatty acids (PUFA). The liberation of AA by PLA2 enzymes sets off a cascade of molecular events that involves downstream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX) metabolites leading to inflammation. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting COX, while Zileuton inhibits LOX and both rely on PLA2 enzymes to provide them with AA. That means PLA2 enzymes can potentially also be targeted to diminish inflammation at an earlier point in the process. In this review we describe extensive efforts reported in the past to define the interactions of PLA2 enzymes with membranes, substrate phospholipids and inhibitors using DXMS, molecular docking, and molecular dynamics (MD) simulations. PMID:26774606

  5. Influence of nanoparticle-membrane electrostatic interactions on membrane fluidity and bending elasticity.

    PubMed

    Santhosh, Poornima Budime; Velikonja, Aljaž; Perutkova, Šarka; Gongadze, Ekaterina; Kulkarni, Mukta; Genova, Julia; Eleršič, Kristina; Iglič, Aleš; Kralj-Iglič, Veronika; Ulrih, Nataša Poklar

    2014-02-01

    The aim of this work is to investigate the effect of electrostatic interactions between the nanoparticles and the membrane lipids on altering the physical properties of the liposomal membrane such as fluidity and bending elasticity. For this purpose, we have used nanoparticles and lipids with different surface charges. Positively charged iron oxide (γ-Fe2O3) nanoparticles, neutral and negatively charged cobalt ferrite (CoFe2O4) nanoparticles were encapsulated in neutral lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine lipid mixture. Membrane fluidity was assessed through the anisotropy measurements using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Though the interaction of both the types of nanoparticles reduced the membrane fluidity, the results were more pronounced in the negatively charged liposomes encapsulated with positively charged iron oxide nanoparticles due to strong electrostatic attractions. X-ray photoelectron spectroscopy results also confirmed the presence of significant quantity of positively charged iron oxide nanoparticles in negatively charged liposomes. Through thermally induced shape fluctuation measurements of the giant liposomes, a considerable reduction in the bending elasticity modulus was observed for cobalt ferrite nanoparticles. The experimental results were supported by the simulation studies using modified Langevin-Poisson-Boltzmann model. PMID:24309194

  6. Interactions between genes involved in exocytotic membrane fusion in paramecium.

    PubMed

    Bonnemain, H; Gulik-Krzywicki, T; Grandchamp, C; Cohen, J

    1992-03-01

    Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9a was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the trichocyst membrane to the plasma membrane and in assembly of the "rosette," a complex array of intramembranous particles in the plasma membrane at the trichocyst insertion sites. No revertants were obtained but two new mutants deficient for rosette assembly were identified, nd16b and nd18, whose gene products appear to interact with that of nd9. Indeed, the double mutants grown at 18 degrees, a permissive temperature for each of the single mutants, are characterized by a deficiency in exocytosis and in rosette assembly, as are also double mutants combining other allelic forms of the same genes. Moreover, aberrant dominance relationships among alleles of nd9 and of nd16 indicate the existence of interactions between identical subunits, which most likely assemble into multimeric structures. The nd16 gene product was shown by microinjection experiments to be a cytosolic factor, as is the nd9 gene product. It is therefore tempting to propose that the nd16 gene product also belongs to the connecting material and is involved in rosette assembly, in cooperation with nd9 and nd18.

  7. Modulation and interactions of charged biomimetic membranes with bivalent ions

    NASA Astrophysics Data System (ADS)

    Kazadi Badiambile, Adolphe

    The biological membrane of an eukaryotic cell is a two-dimensional structure of mostly phospholipids with embedded proteins. This two-dimensional structure plays many key roles in the life of a cell. Transmembrane proteins, for example, play the role of a gate for different ions (such as Ca2+). Also found are peripheral proteins that are used as enzymes for different purposes in the inner leaflet of the plasma membrane. Phospholipids, in particular play three key roles. Firstly, some members of this group are used to store energy. Secondly, the hydrophobic and hydrophilic properties inherent to phospholipids enable them to be used as building blocks of the cell membrane by forming an asymmetric bilayer. This provides a shielding protection against the outer environment while at the same time keeping the organelles and cytosol from leaking out of the cell. Finally lipids are involved in regulating the aggregation of proteins in the membrane. In addition, some subspecies such as phosphatidylinositol (PtdIns) are second messenger molecules in their own right, thus playing an important role in cellular signaling events. In my work presented in this thesis, I am focusing on the role of some phospholipids as signaling molecules and in particular the physicochemical underpinnings that could be used in their spatiotemporal organization in the cellular plasma membrane. I am specifically concerned with the important family of phosphatidylinositol lipids. PtdIns are very well known for their role as signaling molecules in numerous cell events. They are located in the inner leaflet of the plasma membrane as well as part of the membrane of other organelles. Studies of these signaling molecules in their in vivo environment present many challenges: Firstly, the complexity of interactions due to the numerous entities present in eukaryotic cell membranes makes it difficult to establish clear cause and effect relationships. Secondly, due to their size, our inability to probe these

  8. Electrostatic interaction of heterogeneously charged surfaces with semipermeable membranes.

    PubMed

    Maduar, Salim R; Lobaskin, Vladimir; Vinogradova, Olga I

    2013-01-01

    In this paper we study the electrostatic interaction of a heterogeneously charged wall with a neutral semipermeable membrane. The wall consists of periodic stripes, where the charge density varies in one direction. The membrane is in contact with a bulk reservoir of an electrolyte solution and separated from the wall by a thin film of salt-free liquid. One type of ions (small counterions) permeates into the gap. This gives rise to a distance-dependent membrane potential, which translates into a repulsive electrostatic disjoining pressure due to an overlap of counterion clouds in the gap. To quantify it we use two complementary approaches. First, we propose a mean-field theory based on a linearized Poisson-Boltzmann equation and Fourier analysis. These calculations allow us to estimate the effect of a heterogeneous charge pattern at the wall on the induced heterogeneous membrane potential, and the value of the disjoining pressure as a function of the gap. Second, we perform Langevin dynamics simulations of the same system with explicit ions. The results of the two approaches are in good agreement with each other at low surface charges and small gaps, but differ due to nonlinearity at higher charges. These results demonstrate that a heterogeneity of the wall charge can lead to a huge reduction in the electrostatic repulsion, which could dramatically facilitate self-assembly in complex synthetic and biological systems.

  9. Interactions of anesthetics with the membrane-water interface

    NASA Technical Reports Server (NTRS)

    Pohorille, A.; Cieplak, P.; Wilson, M. A.

    1996-01-01

    Although the potency of conventional anesthetics correlates with lipophilicity, an affinity to water also is essential. It was recently found that compounds with very low affinities to water do not produce anesthesia regardless of their lipophilicity. This finding implies that clinical anesthesia might arise because of interactions at molecular sites near the interface of neuronal membranes with the aqueous environment and, therefore, might require increased concentrations of anesthetic molecules at membrane interfaces. As an initial test of this hypothesis, we calculated in molecular dynamics simulations the free energy profiles for the transfer of anesthetic 1,1,2-trifluoroethane and nonanesthetic perfluoroethane across water-membrane and water-hexane interfaces. Consistent with the hypothesis, it was found that trifluoroethane, but not perfluoroethane, exhibits a free energy minimum and, therefore, increased concentrations at both interfaces. The transfer of trifluoroethane from water to the nonpolar hexane or interior of the membrane is accompanied by a considerable, solvent-induced shift in the conformational equilibrium around the C-C bond.

  10. Interactions of membranes with coarse-grain proteins: a comparison

    NASA Astrophysics Data System (ADS)

    Neder, Jörg; Nielaba, Peter; West, Beate; Schmid, Friederike

    2012-12-01

    We study the interactions between lipid bilayers and rigid transmembrane proteins by Monte Carlo simulations of generic coarse-grain models. Different popular protein models are considered and compared with each other, and key parameters such as the hydrophobicity and the hydrophobic mismatch are varied systematically. Furthermore, the properties of the membrane are manipulated by applying different tensions. The response of the membrane to the insertion of single proteins is found to be mostly generic and independent of the choice of the protein model. Likewise, the orientational distributions of single proteins depend mainly on the hydrophobic mismatch and the hydrophobicity of the proteins, and are otherwise similar for all protein models. Orientational distributions are generally found to be very broad, i.e. tilt angles fluctuate very much, in agreement with experimental findings. Weakly hydrophobic proteins respond to positive hydrophobic mismatch by tilting. Strongly hydrophobic (strongly bound) proteins distort the surrounding membrane and tend to remain upright. For proteins with intermediate hydrophobicity, the two mechanisms compete, and as a result, the tilt only sets in if the hydrophobic mismatch exceeds a threshold. Clusters of several strongly hydrophobic proteins with negative positive mismatch may nucleate raft-like structures in membranes. This effect is more pronounced for proteins with rough, structured surfaces.

  11. Characterization of membrane protein interactions by isothermal titration calorimetry.

    PubMed

    Situ, Alan J; Schmidt, Thomas; Mazumder, Parichita; Ulmer, Tobias S

    2014-10-23

    Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of -4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500±100s(-1) and 2.1±0.1s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.

  12. Benchmarking of Force Fields for Molecule-Membrane Interactions.

    PubMed

    Paloncýová, Markéta; Fabre, Gabin; DeVane, Russell H; Trouillas, Patrick; Berka, Karel; Otyepka, Michal

    2014-09-01

    Studies of drug-membrane interactions witness an ever-growing interest, as penetration, accumulation, and positioning of drugs play a crucial role in drug delivery and metabolism in human body. Molecular dynamics simulations complement nicely experimental measurements and provide us with new insight into drug-membrane interactions; however, the quality of the theoretical data dramatically depends on the quality of the force field used. We calculated the free energy profiles of 11 molecules through a model dimyristoylphosphatidylcholine (DMPC) membrane bilayer using five force fields, namely Berger, Slipids, CHARMM36, GAFFlipids, and GROMOS 43A1-S3. For the sake of comparison, we also employed the semicontinuous tool COSMOmic. High correlation was observed between theoretical and experimental partition coefficients (log K). Partition coefficients calculated by all-atomic force fields (Slipids, CHARMM36, and GAFFlipids) and COSMOmic differed by less than 0.75 log units from the experiment and Slipids emerged as the best performing force field. This work provides the following recommendations (i) for a global, systematic and high throughput thermodynamic evaluations (e.g., log K) of drugs COSMOmic is a tool of choice due to low computational costs; (ii) for studies of the hydrophilic molecules CHARMM36 should be considered; and (iii) for studies of more complex systems, taking into account all pros and cons, Slipids is the force field of choice.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  14. Membrane interactions between secretion granules and plasmalemma in three exocrine glands.

    PubMed

    Tanaka, Y; De Camilli, P; Meldolesi, J

    1980-02-01

    significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis. PMID:7380885

  15. Membrane Assays to Characterize Interaction of Drugs with ABCB1.

    PubMed

    Fekete, Zsolt; Rajnai, Zsuzsanna; Nagy, Tünde; Jakab, Katalin Tauberné; Kurunczi, Anita; Gémes, Katalin; Herédi-Szabó, Krisztina; Fülöp, Ferenc; Tóth, Gábor K; Czerwinski, Maciej; Loewen, Greg; Krajcsi, Peter

    2015-12-01

    ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions. PMID:25926125

  16. Interaction of two different types of membrane proteins with model membranes investigated by FTIR ATR spectroscopy

    NASA Astrophysics Data System (ADS)

    Siam, M.; Reiter, G.; Schwarzott, M.; Baurecht, D.; Fringeli, U. P.

    1998-06-01

    Polarized FTIR ATR spectroscopy was used to investigate the interaction of mitochondrial creatine kinase (Mi-CK) and intestinal alkaline phosphatase (AP) with model membrane assemblies. Mi-CK was immobilized by adsorption to the negatively charged cardiolipin (CL) leaflet of a supported CL/DPPA bilayer. H-D-exchange of the enzyme and the stability under flowthrough conditions of the protein/membrane assembly were examined. AP, however, was bound to a DPPA Langmuir-Blodgett layer (LBL), followed by the completion of a bilayer-like structure by adsorption of POPC molecules from a vesicular solution. It turned out that the POPC adsorbate exhibited decreased molecular order compared to the POPC molecules on a supported POPC/DPPA bilayer. Enzymatic activity of immobilized AP was determined with p-nitrophenyl phosphate (p-NPP) as substrate and remained unchanged for at least 2 days.

  17. Studies on the interactions between parabens and lipid membrane components in monolayers at the air/aqueous solution interface.

    PubMed

    Flasiński, Michał; Gawryś, Maciej; Broniatowski, Marcin; Wydro, Paweł

    2016-04-01

    The interactions between parabens (PBs) and lipid components of mammalian and bacterial cell membranes were investigated in model systems of Langmuir monolayers. Me-, Et-, Pr- and Bu-paraben studied in this paper are frequently applied as cosmetics and food preservatives, since they possess broad antimicrobial activity. The mode of PB action is connected with their incorporation into the membrane of bacterial organisms, however; it is not known what is the role of the respective lipid species in this mechanism. This problem is crucial to understand the differences in paraben activity toward individual microorganisms and to shed the light onto the problem of PB cytotoxicity reported in studies on mammalian cells. In this paper, the mentioned aspects were investigated with application of the Langmuir monolayer technique complemented with BAM and GIXD. Our experiments revealed that the influence of PBs depends on their chemical structure, solution concentration and on the class of lipid. The strongest modification of the monolayer characteristics, leading to its collapse at low surface pressure, occurred in the presence of BuPB, having the largest chain. PBs interact preferentially with the monolayers possessing low degree of condensation, whereas for LC state, the effect was weaker and observed only as modification of the 2D unit cells. In the model systems, PBs interact with phospholipids characteristic for mammalian membranes (phosphatidylcholine) stronger than with bacterial (phosphatidylglycerol and cardiolipin). This strong influence of parabens on the model systems composed of animal lipids may explain cytotoxic activity of these preservatives. PMID:26777770

  18. Studies on the interactions between parabens and lipid membrane components in monolayers at the air/aqueous solution interface.

    PubMed

    Flasiński, Michał; Gawryś, Maciej; Broniatowski, Marcin; Wydro, Paweł

    2016-04-01

    The interactions between parabens (PBs) and lipid components of mammalian and bacterial cell membranes were investigated in model systems of Langmuir monolayers. Me-, Et-, Pr- and Bu-paraben studied in this paper are frequently applied as cosmetics and food preservatives, since they possess broad antimicrobial activity. The mode of PB action is connected with their incorporation into the membrane of bacterial organisms, however; it is not known what is the role of the respective lipid species in this mechanism. This problem is crucial to understand the differences in paraben activity toward individual microorganisms and to shed the light onto the problem of PB cytotoxicity reported in studies on mammalian cells. In this paper, the mentioned aspects were investigated with application of the Langmuir monolayer technique complemented with BAM and GIXD. Our experiments revealed that the influence of PBs depends on their chemical structure, solution concentration and on the class of lipid. The strongest modification of the monolayer characteristics, leading to its collapse at low surface pressure, occurred in the presence of BuPB, having the largest chain. PBs interact preferentially with the monolayers possessing low degree of condensation, whereas for LC state, the effect was weaker and observed only as modification of the 2D unit cells. In the model systems, PBs interact with phospholipids characteristic for mammalian membranes (phosphatidylcholine) stronger than with bacterial (phosphatidylglycerol and cardiolipin). This strong influence of parabens on the model systems composed of animal lipids may explain cytotoxic activity of these preservatives.

  19. Quantitative assessment of interfacial interactions with rough membrane surface and its implications for membrane selection and fabrication in a MBR.

    PubMed

    Chen, Jianrong; Mei, Rongwu; Shen, Liguo; Ding, Linxian; He, Yiming; Lin, Hongjun; Hong, Huachang

    2015-03-01

    The interfacial interactions between a foulant particle and rough membrane surface in a submerged membrane bioreactor (MBR) were quantitatively assessed by using a new-developed method. It was found that the profile of total interaction versus separation distance was complicated. There were an energy barrier and two negative energy ranges in the profile. Further analysis showed that roughness scale significantly affected the strength and properties of interfacial interactions. It was revealed that there existed a critical range of roughness scale within which the total energy in the separation distance ranged from 0 to several nanometers was continually repulsive. Decrease in foulant size would increase the strength of specific interaction energy, but did not change the existence of a critical roughness scale range. These findings suggested the possibility to "tailor" membrane surface morphology for membrane fouling mitigation, and thus gave significant implications for membrane selection and fabrication in MBRs. PMID:25553567

  20. Calorimetric evidence of interaction of brominated flame retardants with membrane model.

    PubMed

    Librando, Vito; Accolla, Maria Lorena; Minniti, Zelica; Pappalardo, Matteo; Castelli, Francesco; Cascio, Orazio; Sarpietro, Maria Grazia

    2015-05-01

    The presence of polybrominated flame retardants in the environment seems to be increasing in the past decade. Considering the toxic effects of these pollutants, it is important evaluating the potential interaction with biological membranes for a risk assessment. In this study low and high brominated biphenyls and biphenyl ethers were used to investigate their interaction with biological membrane models constituted by liposomes, using differential scanning calorimetry (DSC) technique. The medium influence on membrane absorption was also assessed. The findings indicate that membrane interaction is controlled by compound structural characteristics. The membrane absorption is allowed by lipophilic medium; instead hydrophilic medium prevents membrane permeation.

  1. On the interaction of the liposomal membrane with blood components.

    PubMed

    Miller, I F; Hoag, J M; Rooney, M W

    1992-01-01

    Liposome-encapsulated hemoglobin (LEH) has been shown to be a viable candidate as a blood replacement. However, few data have been presented as to how LEH interacts with normal blood components. Liposomes were prepared from egg lecithin, cholesterol, and dicetyl phosphate or phosphatidic acid, and mixed with fresh blood plasma or whole blood. Erythrocyte osmotic fragility, prothrombin time (extrinsic coagulation efficiency), activated partial thromboplastin time (intrinsic coagulation efficiency), plasma clot stability in urea (fibrin stabilizing factor), and clot retraction (platelet activation) were measured. Although liposomes were found to bind extensively to erythrocytes, all tests indicated that the liposomes had no significant adverse effects, provided that normal levels of plasma Ca++ were maintained. The ability of liposomes to absorb Ca++ from the plasma was related directly to the amount of dicetyl phosphate or phosphatidic acid present and thus, presumably, to the presence of negatively charged species in the membrane. The mechanics of deformation of the LEH membrane were investigated by encapsulating Hemoglobin S in liposomes. Liposomes containing Hemoglobin S were found to sickle when deoxygenated, but not liposomes containing normal hemoglobin. Shape analysis of sickled liposomes yielded a deforming stress of 10(6) dynes/cm2, about 50 times greater than the reported limit for shear elasticity of the erythrocyte membrane. PMID:1391486

  2. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    PubMed Central

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  3. Multitasking water-soluble synthetic G-quartets: from preferential RNA-quadruplex interaction to biocatalytic activity.

    PubMed

    Haudecoeur, Romain; Stefan, Loic; Monchaud, David

    2013-09-16

    Natural G-quartets, a cyclic and coplanar array of four guanine residues held together through a Watson-Crick/Hoogsteen hydrogen-bond network, have received recently much attention due to their involvement in G-quadruplex DNA, an alternative higher-order DNA structure strongly suspected to play important roles in key cellular events. Besides this, synthetic G-quartets (SQ), which artificially mimic native G-quartets, have also been widely studied for their involvement in nanotechnological applications (i.e., nanowires, artificial ion channels, etc.). In contrast, intramolecular synthetic G-quartets (iSQ), also named template-assembled synthetic G-quartets (TASQ), have been more sparingly investigated, despite a technological potential just as interesting. Herein, we report on a particular iSQ named (PNA) DOTASQ, which demonstrates very interesting properties in terms of DNA and RNA interaction (notably its selective recognition of quadruplexes according to a bioinspired process) and catalytic activities, through its ability to perform peroxidase-like hemin-mediated oxidations either in an autonomous fashion (i.e., as pre-catalyst for TASQzyme reactions) or in conjunction with quadruplex DNA (i.e., as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applications.

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

    PubMed

    Pink, D A; Chapman, D

    1979-04-01

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

  5. Solute-solvent and solvent-solvent interactions in the preferential solvation of 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide in 24 binary solvent mixtures

    NASA Astrophysics Data System (ADS)

    Bevilaqua, Tharly; Gonçalves, Thaini F.; Venturini, Cristina de G.; Machado, Vanderlei G.

    2006-11-01

    The molar transition energy ( ET) polarity values for the dye 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide were collected in binary mixtures comprising a hydrogen-bond accepting (HBA) solvent (acetone, acetonitrile, dimethyl sulfoxide (DMSO), and N, N-dimethylformamide (DMF)) and a hydrogen-bond donating (HBD) solvent (water, methanol, ethanol, propan-2-ol, and butan-1-ol). Data referring to mixtures of water with alcohols were also analyzed. These data were used in the study of the preferential solvation of the probe, in terms of both solute-solvent and solvent-solvent interactions. These latter interactions are of importance in explaining the synergistic behavior observed for many mixed solvent systems. All data were successfully fitted to a model based on solvent-exchange equilibria. The ET values of the dye dissolved in the solvents show that the position of the solvatochromic absorption band of the dye is dependent on the medium polarity. The solvation of the dye in HBA solvents occurs with a very important contribution from ion-dipole interactions. In HBD solvents, the hydrogen bonding between the dimethylamino group in the dye and the OH group in the solvent plays an important role in the solvation of the dye. The interaction of the hydroxylic solvent with the other component in the mixture can lead to the formation of hydrogen-bonded complexes, which solvate the dye using a lower polar moiety, i.e. alkyl groups in the solvents. The dye has a hydrophobic nature and a dimethylamino group with a minor capability for hydrogen bonding with the medium in comparison with the phenolate group present in Reichardt's pyridiniophenolate. Thus, the probe is able to detect solvent-solvent interactions, which are implicit to the observed synergistic behavior.

  6. α-Cyclodextrin Interacts Close to Vinblastine Site of Tubulin and Delivers Curcumin Preferentially to the Tubulin Surface of Cancer Cell.

    PubMed

    Jana, Batakrishna; Mohapatra, Saswat; Mondal, Prasenjit; Barman, Surajit; Pradhan, Krishnangsu; Saha, Abhijit; Ghosh, Surajit

    2016-06-01

    Tubulin is the key cytoskeleton component, which plays a crucial role in eukaryotic cell division. Many anticancer drugs have been developed targeting the tubulin surface. Recently, it has been shown that few polyhydroxy carbohydrates perturb tubulin polymerization. Cyclodextrin (CD), a polyhydroxy carbohydrate, has been extensively used as the delivery vehicle for delivery of hydrophobic drugs to the cancer cell. However, interaction of CD with intracellular components has not been addressed before. In this Article, we have shown for the first time that α-CD interacts with tubulin close to the vinblastine site using molecular docking and Förster resonance energy transfer (FRET) experiment. In addition, we have shown that α-CD binds with intracellular tubulin/microtubule. It delivers a high amount of curcumin onto the cancer cell, which causes severe disruption of intracellular microtubules. Finally, we have shown that the inclusion complex of α-CD and curcumin (CCC) preferentially enters into the human lung cancer cell (A549) as compared to the normal lung fibroblast cell (WI38), causes apoptotic death, activates tumor suppressor protein (p53) and cyclin-dependent kinase inhibitor 1 (p21), and inhibits 3D spheroid growth of cancer cell. PMID:27228201

  7. Tissue Factor Residues That Putatively Interact with Membrane Phospholipids

    PubMed Central

    Ke, Ke; Yuan, Jian; Morrissey, James H.

    2014-01-01

    Blood clotting is initiated by the two-subunit enzyme consisting of the plasma protease, factor VIIa (the catalytic subunit), bound to the integral membrane protein, tissue factor (the regulatory subunit). Molecular dynamics simulations have predicted that certain residues in the tissue factor ectodomain interact with phosphatidylserine headgroups to ensure optimal positioning of the tissue factor/factor VIIa complex relative to its membrane-bound protein substrates, factors IX and X. In this study, we individually mutated to alanine all the putative phosphatidylserine-interactive residues in the tissue factor ectodomain and measured their effects on tissue factor cofactor function (activation of factors IX and X by tissue factor/factor VIIa, and clotting of plasma). Some tissue factor mutants exhibited decreased activity in all three assays, with the most profound defects observed from mutations in or near the flexible loop from Lys159 to Gly164. The decreased activity of all of these tissue factor mutants could be partially or completely overcome by increasing the phosphatidylserine content of tissue factor-liposomes. Additionally, yeast surface display was used to screen a random library of tissue factor mutants for enhanced factor VIIa binding. Surprisingly, mutations at a single amino acid (Lys165) predominated, with the Lys165→Glu mutant exhibiting a 3-fold enhancement in factor VIIa binding affinity. Our studies reveal the functional contributions of residues in the C-terminal half of the tissue factor ectodomain that are implicated in interacting with phosphatidylserine headgroups to enhance tissue factor cofactor activity, possibly by allosterically modulating the conformation of the adjacent substrate-binding exosite region of tissue factor. PMID:24516673

  8. Mixed matrix membranes with strengthened MOFs/polymer interfacial interaction and improved membrane performance.

    PubMed

    Lin, Rijia; Ge, Lei; Hou, Lei; Strounina, Ekaterina; Rudolph, Victor; Zhu, Zhonghua

    2014-04-23

    MOFs-based mixed matrix membranes (MMMs) have attracted extensive attention in recent years due to their potential high separation performance, the low cost, and good mechanical properties. However, it is still very challenging to achieve defect-free interface between micrometer-sized MOFs and a polymer matrix. In this study, [Cd2L(H2O)]2·5H2O (Cd-6F) synthesized using 4,4'-(hexafluoroisopropylidene)diphthalic anhydride (6FDA) as an organic ligand was introduced into the 6FDA-ODA polyimide matrix to achieve novel MOF MMMs. A specific interfacial interaction between MOF crystals and polymer chains was innovatively targeted and achieved through in situ polymerization procedure. The enhanced adhesion between MOF particles and polymer phase was observed, and the improved interfacial interaction between Cd-6F and the 6FDA-ODA polyimide matrix was confirmed by detailed characterizations including FTIR and NMR. In the meantime, the gas permeance and selectivity of the MMMs are strongly dependent on their morphology. The MMM derived from in situ polymerization presents excellent interfaces between micrometer-sized MOF crystals and the polymer matrix, resulting in increased permeability and selectivity. The strategy shown here can be further utilized to select the MOF/polymer pair, eliminate interfacial voids, and improve membrane separation performance of MOFs-based MMMs.

  9. Role of Lipid Composition on the Interaction between a Tryptophan-Rich Protein and Model Bacterial Membranes.

    PubMed

    Sanders, Michael R; Clifton, Luke A; Frazier, Richard A; Green, Rebecca J

    2016-03-01

    The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements, and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition, and fluidity on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity. PMID:26813886

  10. Interaction of amphiphilic molecules with biological membranes. A model for nonspecific and specific drug effects with membranes.

    PubMed

    Herbette, L; Napolitano, C A; Messineo, F C; Katz, A M

    1985-01-01

    The nonspecific interactions of propranolol, timolol, and ethanol with model and sarcoplasmic reticulum membranes were determined utilizing radioisotopic association differential scanning calorimetry, and neutron diffraction. Differential scanning calorimetry performed on mixtures of these amphiphilic compounds and model membrane bilayers composed of dimyristoyllecithin showed that propranolol was approximately 25 times more lipid-soluble than timolol and at least 100 times more lipid-soluble than ethanol. Neutron diffraction showed that the solvation of propranolol was within the fatty acyl chain region of the lipid bilayer. This solvation correlated with the effect of propranolol to inhibit ATP-dependent calcium transport in isolated rabbit skeletal muscle sarcoplasmic reticulum, a membrane that lacks beta-adrenergic receptors. In contrast, the major site of interaction of ethanol was within the aqueous compartment hydrating the sarcoplasmic reticulum membrane. A model for nonspecific drug interaction with the sarcoplasmic reticulum membrane based on the site of interaction of these amphiphiles and their relative potencies to inhibit calcium transport by these membranes is proposed. In principle, this model could be extended to specific drug interactions with membranes.

  11. Similarities and differences of serotonin and its precursors in their interactions with model membranes studied by molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Wood, Irene; Martini, M. Florencia; Pickholz, Mónica

    2013-08-01

    In this work, we report a molecular dynamics (MD) simulations study of relevant biological molecules as serotonin (neutral and protonated) and its precursors, tryptophan and 5-hydroxy-tryptophan, in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC). The simulations were carried out at the fluid lamellar phase of POPC at constant pressure and temperature conditions. Two guest molecules of each type were initially placed at the water phase. We have analyzed, the main localization, preferential orientation and specific interactions of the guest molecules within the bilayer. During the simulation run, the four molecules were preferentially found at the water-lipid interphase. We found that the interactions that stabilized the systems are essentially hydrogen bonds, salt bridges and cation-π. None of the guest molecules have access to the hydrophobic region of the bilayer. Besides, zwitterionic molecules have access to the water phase, while protonated serotonin is anchored in the interphase. Even taking into account that these simulations were done using a model membrane, our results suggest that the studied molecules could not cross the blood brain barrier by diffusion. These results are in good agreement with works that show that serotonin and Trp do not cross the BBB by simple diffusion.

  12. A new method for modeling rough membrane surface and calculation of interfacial interactions.

    PubMed

    Zhao, Leihong; Zhang, Meijia; He, Yiming; Chen, Jianrong; Hong, Huachang; Liao, Bao-Qiang; Lin, Hongjun

    2016-01-01

    Membrane fouling control necessitates the establishment of an effective method to assess interfacial interactions between foulants and rough surface membrane. This study proposed a new method which includes a rigorous mathematical equation for modeling membrane surface morphology, and combination of surface element integration (SEI) method and the composite Simpson's approach for assessment of interfacial interactions. The new method provides a complete solution to quantitatively calculate interfacial interactions between foulants and rough surface membrane. Application of this method in a membrane bioreactor (MBR) showed that, high calculation accuracy could be achieved by setting high segment number, and moreover, the strength of three energy components and energy barrier was remarkably impaired by the existence of roughness on the membrane surface, indicating that membrane surface morphology exerted profound effects on membrane fouling in the MBR. Good agreement between calculation prediction and fouling phenomena was found, suggesting the feasibility of this method.

  13. Cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes.

    PubMed

    Arsov, Zoran; Nemec, Marjana; Schara, Milan; Johansson, Henrik; Langel, Ulo; Zorko, Matjaz

    2008-12-01

    Interaction of the cell-penetrating peptide (CPP) cysteine-transportan (Cys-TP) with model lipid membranes was examined by spin-label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys-TP on membrane structure was studied. The influence of Cys-TP on membrane permeability was monitored by the reduction of a liposome-trapped water-soluble spin probe. Cys-TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys-TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so-called liquid-ordered phase, no effect of Cys-TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys-TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP-lipid interaction in laterally heterogeneous membranes, while it implies that the CPP-lipid interaction can be different at different points along the membrane. PMID:18683276

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

    PubMed

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

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26940626

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

    PubMed

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

    2016-07-01

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

  16. Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids

    NASA Technical Reports Server (NTRS)

    Lacey, J. C. Jr; Wickramasinghe, N. S.; Sabatini, R. S.

    1992-01-01

    We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

  17. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    PubMed Central

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains. PMID:12803918

  18. Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

    PubMed Central

    Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

    1998-01-01

    The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

  19. Interaction of a peptide nanotube with a water membrane interface

    NASA Astrophysics Data System (ADS)

    Chipot, Christophe; Tarek, Mounir

    2006-03-01

    Inserting peptide nanotubes into lipid bilayers modulates the permeability properties of the cell wall, thus conferring potential bacteriocidal capability. Interaction of a peptide nanotube formed by eight cyclo[RRKWLWLW] subunits with the surface of a hydrated dimyristoylphosphatidylcholine bilayer is investigated using molecular dynamics simulations. The present sequence of alternated D-L-α-amino acids has been shown to yield remarkable antibacterial in vitro activity, and the chosen topoisomer corresponds to the optimum amphipathy of the tubular structure, whereby non-polar and charged side chains are segregated by the aqueous interface. The cohesion of the nanotube is ensured by a scaffold of intermolecular hydrogen bonds between adjacent cyclic peptides, supplemented by favorable like-charged contacts of arginine side chains. It is further reinforced by interactions of charged residues with the lipid head groups and of non-polar residues with the lipid acyl chains. The simulation reveals a partial breaking of the synthetic channel accompanying its early insertion into the lipid bilayer. The latter opens new questions about how peptide nanotubes permeate the membrane, in particular whether or not (i) self-assembly precedes partitioning and (ii) translocation occurs with the complete tubular structure.

  20. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Halpert, James R.

    2015-01-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  1. Molecular Interaction between Magainin 2 and Model Membranes in Situ

    PubMed Central

    Nguyen, Khoi; Le Clair, Stéphanie V.; Ye, Shuji; Chen, Zhan

    2009-01-01

    In this paper, we investigated the molecular interactions of Magainin 2 with model cell membranes using Sum Frequency Generation (SFG) vibrational spectroscopy and Attenuated Total Reflectance – Fourier Transform Infrared spectroscopy (ATR-FTIR). Symmetric 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (POPG) and 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) bilayers, which model the bacterial and mammalian cell membranes respectively, were used in the studies. It was observed by SFG that Magainin 2 orients relatively parallel to the POPG lipid bilayer surface at low solution concentrations, around 200 nM. When increasing the Magainin 2 concentration to 800 nM, both SFG and ATR-FTIR results indicate that Magainin 2 molecules insert into the POPG bilayer and adopt a transmembrane orientation with an angle of about 20 degrees from the POPG bilayer normal. For the POPC bilayer, even at a much higher peptide concentration of 2.0 µM, no ATR-FTIR signal was detected. For this concentration on POPC, SFG studies indicated that Magainin 2 molecules adopt an orientation nearly parallel to the bilayer surface, with an orientation angle of 75 degrees from the surface normal. This shows that SFG has a much better detection limit than ATR-FTIR and can therefore be applied to study interfacial molecules with much lower surface coverage. This Magainin 2 orientation study and further investigation of the lipid bilayer SFG signals support the proposed toroidal pore model for the antimicrobial activity of Magainin 2. PMID:19728722

  2. Electrospun polyvinyl alcohol-polyvinyl pyrrolidone nanofibrous membranes for interactive wound dressing application.

    PubMed

    Shankhwar, Nisha; Kumar, Manishekhar; Mandal, Biman B; Robi, P S; Srinivasan, A

    2016-01-01

    Cross-linked polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) composite nanofibrous membranes have been prepared by electrospinning. Mechanical properties of the membranes improved significantly with PVP addition. PVP improved hydrophilicity and sustainable degradation of the membranes. Biocompatibility of the membranes was assessed by in vitro culture of native skin cells (L929 fibroblast and HaCaT keratinocytes). Tests showed sustained release of the antibiotic ciprofloxacin hydrochloride monohydrate by the membranes. Further, zone of inhibition study against Staphylococcus aureus growth demonstrated protective action against external pathogenic microbes. These studies show these simple PVA-PVP nanofibrous membranes are promising interactive antibiotic-eluting wound dressing materials.

  3. Amyloid-β peptides in interaction with raft-mime model membranes: a neutron reflectivity insight.

    PubMed

    Rondelli, Valeria; Brocca, Paola; Motta, Simona; Messa, Massimo; Colombo, Laura; Salmona, Mario; Fragneto, Giovanna; Cantù, Laura; Del Favero, Elena

    2016-01-01

    The role of first-stage β-amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β-amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment. PMID:26880066

  4. The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

    PubMed

    Ho, Ruoya; Stroupe, Christopher

    2016-10-01

    Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. PMID:27307091

  5. The interactions of squalene, alkanes and other mineral oils with model membranes; effects on membrane heterogeneity and function.

    PubMed

    Richens, Joanna L; Lane, Jordan S; Mather, Melissa L; O'Shea, Paul

    2015-11-01

    Droplet interface bilayers (DIBs) offer many favourable facets as an artificial membrane system but the influence of any residual oil that remains in the bilayer following preparation is ill-defined. In this study the fluorescent membrane probes di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (Di-8-ANEPPS) and Fluoresceinphosphatidylethanolamine (FPE) were used to help understand the nature of the phospholipid-oil interaction and to examine any structural and functional consequences of such interactions on membrane bilayer properties. Concentration-dependent modifications of the membrane dipole potential were found to occur in phospholipid vesicles exposed to a variety of different oils. Incorporation of oil into the lipid bilayer was shown to have no significant effect on the movement of fatty acids across the lipid bilayer. Changes in membrane heterogeneity were, however, demonstrated with increased microdomain formation being visible in the bilayer following exposure to mineral oil, pentadecane and squalene. As it is important that artificial systems provide an accurate representation of the membrane environment, careful consideration should be taken prior to the application of DIBs in studies of membrane structure and organisation.

  6. Nonbonded interactions in membrane active cyclic biopolymers. IV - Cation dependence

    NASA Technical Reports Server (NTRS)

    Radhakrishnan, R.; Srinivasan, S.; Prasad, C. V.; Brinda, S. R.; Macelroy, R. D.; Sundaram, K.

    1980-01-01

    Interactions of valinomycin and form of its analogs in several conformations with the central ions Li(+), Na(+), K(+), Rb(+) and Cs(+) are investigated as part of a study of the specific preference of valinomycin for potassium and the mechanisms of carrier-mediated ion transport across membranes. Ion binding energies and conformational potential energies are calculated taking into account polarization energy formulas and repulsive energy between the central ion and the ligand atoms for conformations representing various stages in ion capture and release for each of the two ring chiralities of valinomycin and its analogs. Results allow the prediction of the chirality and conformation most likely to be observed for a given analog, and may be used to synthesize analogs with a desired rigidity or flexibility. The binding energies with the alkali metal cations are found to decrease with increasing ion size, and to be smaller than the corresponding ion hydration energies. It is pointed out that the observed potassium preference may be explainable in terms of differences between binding and hydration energies. Binding energies are also noted to depend on ligand conformation.

  7. Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

    PubMed Central

    Guan, Yan; Shan, Xiaonan; Zhang, Fenni; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-01

    Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell. PMID:26601298

  8. The effect of solute-membrane interaction on solute permeation under supersaturated conditions.

    PubMed

    Zhang, Jingsi; Sun, Mingjing; Fan, Aiping; Wang, Zheng; Zhao, Yanjun

    2013-01-30

    The purpose of this work was to investigate the effect of solute-membrane interaction under supersaturated conditions on the transport of model solute (salicylic acid) across poly(dimethylsiloxane) (PDMS) membrane. Supersaturated systems with a degree of saturation (DS) up to 8 were prepared using a molecular form technique with water as the vehicle to minimize the vehicle-membrane interaction. The spectroscopic and thermal analysis revealed the presence of both hydrogen bonding and nonpolar interaction between the solute and PDMS. Upon treatment by supersaturated solutions the degree of solute-membrane interaction increased with increasing DS. This enhanced the barrier property of PDMS and thus led to the flux attenuation compared to that calculated by Higuchi equation. This work highlighted the importance of solute-membrane interaction under supersaturation in the flux reduction, which should be considered when designing, and optimizing supersaturated topical and transdermal drug delivery systems.

  9. Membrane Interaction of Antimicrobial Peptides Using E. coli Lipid Extract as Model Bacterial Cell Membranes and SFG Spectroscopy

    PubMed Central

    Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan

    2015-01-01

    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/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. PMID:25707312

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

    PubMed

    Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan

    2015-04-01

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

  11. Interaction between bending and tension forces in bilayer membranes.

    PubMed Central

    Secomb, T W

    1988-01-01

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

  12. Controlled CO preferential oxidation

    DOEpatents

    Meltser, M.A.; Hoch, M.M.

    1997-06-10

    Method is described for controlling the supply of air to a PROX (PReferential OXidation for CO cleanup) reactor for the preferential oxidation in the presence of hydrogen wherein the concentration of the hydrogen entering and exiting the PROX reactor is monitored, the difference there between correlated to the amount of air needed to minimize such difference, and based thereon the air supply to the PROX reactor adjusted to provide such amount and minimize such difference. 2 figs.

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

    PubMed

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

    1978-06-16

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

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

    PubMed Central

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

    2009-01-01

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

  15. MAPS: an interactive web server for membrane annotation of transmembrane protein structures.

    PubMed

    Cheema, Jitender; Basu, Gautam

    2011-04-01

    The exact positioning of the membrane in transmembrane (TM) proteins plays important functional roles. Yet, the structures of TM proteins in protein data bank (pdb) have no information about the explicit position of the membrane. Using a simple hydrophobic lipid-protein mismatch energy function and a flexible lipid/water boundary, the position of lipid bilayer for representative TM proteins in pdb have been annotated. A web server called MAPS (Membrane Annotation of Protein Structures; available at: http://www.boseinst.ernet.in/gautam/maps) has been set up that allows the user to interactively analyze membrane-protein orientations of any uploaded pdb structure with user-defined membrane flexibility parameters.

  16. Weakly Stable Regions and Protein-Protein Interactions in Beta-Barrel Membrane Proteins

    PubMed Central

    Naveed, Hammad; Liang, Jie

    2014-01-01

    We briefly discuss recent progress in computational characterization of the sequence and structural properties of β-barrel membrane properties. We discuss the emerging concept of weakly stable regions in β-barrel membrane proteins, computational methods to identify these regions and mechanisms adopted by β-barrel membrane proteins in nature to stabilize them. We further discuss computational methods to identify protein-protein interactions in β-barrel membrane proteins and recent experimental studies that aim at altering the biophysical properties including oligomerization state and stability of β-barrel membrane proteins based on the emerging organization principles of these proteins from recent computational studies. PMID:23713778

  17. Effect of lipid head group interactions on membrane properties and membrane-induced cationic β-hairpin folding.

    PubMed

    Ganesan, Sai J; Xu, Hongcheng; Matysiak, Silvina

    2016-07-21

    Stages in POPS membrane induced SVS-1 folding. One key characteristic of mIFs is the dielectric gradient and subsequently, electrostatic potential that arises from dipolar interactions in the head group region. In this work, we present a coarse-grained (CG) model for anionic and zwitterionic lipids that accounts for dipolar intricacies in the head group region. Prior work on adding dipolar interactions in a coarse grained (CG) model for peptides enabled us to achieve α/β secondary structure content de novo, without any added bias. We have now extended this idea to lipids. To mimic dipolar interactions, two dummy particles with opposite charges are added to CG polar beads. These two dummy charges represent a fluctuating dipole that introduces structural polarization into the head group region. We have used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine) as our model lipids. We characterize structural, dynamic, and dielectric properties of our CG bilayer, along with the effect of monovalent ions. We observe head group dipoles to play a significant role in membrane dielectric gradient and lipid clustering induced by dipole-dipole interactions in POPS lipids. In addition, we studied membrane-induced peptide folding of a cationic antimicrobial peptide with anticancer activity, SVS-1. We find that membrane-induced peptide folding is driven by both (a) cooperativity in peptide self-interaction and (b) cooperativity in membrane-peptide interaction. In particular, dipolar interactions between the peptide backbone and lipid head groups contribute to stabilizing folded conformations. PMID:27165814

  18. Wrapping of a deformable nanoparticle by the cell membrane: Insights into the flexibility-regulated nanoparticle-membrane interaction

    NASA Astrophysics Data System (ADS)

    Tang, Huayuan; Zhang, Hongwu; Ye, Hongfei; Zheng, Yonggang

    2016-09-01

    Although many researches have been conducted on the interaction of the cell membrane with the rigid nanoparticle (NP), relatively little is known about the interaction of the membrane with the deformable NP, which is a promising kind of drug delivery carrier. In this paper, we investigate the wrapping of a deformable NP by the membrane, with particular attention paid to the location of the NP. Phase diagrams with respect to the normalized NP-membrane adhesion strength and the bending stiffness ratio between the NP and membrane are presented. The results show that the NP is easier to be fully wrapped but harder to be shallowly wrapped when the NP locates outside than inside the vesicle. For the system with an outside NP, there are three distinct stages separated by two critical bending stiffness ratios as the NP becomes softer. Moreover, the critical normalized adhesion strength required for a deformable NP to be fully wrapped is the same as that for a rigid NP when the bending stiffness ratio is higher than a critical value, which is different from the wrapping behavior by an initially flat membrane. In addition, a larger vesicle size facilitates the full wrapping configuration when the NP is inside, whereas it prohibits it when the NP is outside. These results are consistent with the previous research and can provide guidelines for the design of drug delivery systems based on the flexibility-tunable NPs.

  19. Membrane IgD-positive B cells of "low-IgD serum phenotype" individuals fail to secrete IgD and fail to shift to preferential lambda light-chain expression in vitro.

    PubMed

    Litwin, S D; Zehr, B D

    1987-03-01

    IgD production by short-term human peripheral blood mononuclear cell (PBM) cultures was studied to establish the in vitro correlates of low serum IgD expression. Cells of persons with less than 3 micrograms/ml IgD in the serum, referred to as the low-serum IgD phenotype (LISP), were analyzed. Advantage was taken of recently developed data on spontaneous IgD biosynthesis by human B cells and the observation that lambda light chains are preferentially expressed by IgD-secreting cells in vitro. Initial analysis of an IgD serum distribution showed that all LISP sera contained low but detectable amounts of IgD, with a mean value of 0.85 microgram/ml; this figure was 30- to 35-fold lower than the mean of the majority of the population. LISP PBM contained normal numbers of IgD-positive B cells which displayed a normal intensity of IgD per cell using comparative analysis of mean channel fluorescence by cell flow cytometry. Several lines of evidence suggested that IgD-secreting cells could not be generated from LISP lymphocytes in vitro. Namely, it was found that no IgD immunoglobulin-containing cells were found among PBM of LISP persons; cell lysates enriched for the intracellular fraction by Triton X-114 phase separation showed low IgD in LISP cells despite "normal" amounts of IgD in membrane-enriched fraction preparations; there was no spontaneous IgD secretion by any LISP PBM cultures; and neither LISP sera nor cellular IgD preparations showed IgD lambda/kappa ratios greater than 1.0, indicative of the absence of the preferential lambda light-chain expression associated with secretion of IgD.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3106400

  20. Computational models for predicting interactions with membrane transporters.

    PubMed

    Xu, Y; Shen, Q; Liu, X; Lu, J; Li, S; Luo, C; Gong, L; Luo, X; Zheng, M; Jiang, H

    2013-01-01

    Membrane transporters, including two members: ATP-binding cassette (ABC) transporters and solute carrier (SLC) transporters are proteins that play important roles to facilitate molecules into and out of cells. Consequently, these transporters can be major determinants of the therapeutic efficacy, toxicity and pharmacokinetics of a variety of drugs. Considering the time and expense of bio-experiments taking, research should be driven by evaluation of efficacy and safety. Computational methods arise to be a complementary choice. In this article, we provide an overview of the contribution that computational methods made in transporters field in the past decades. At the beginning, we present a brief introduction about the structure and function of major members of two families in transporters. In the second part, we focus on widely used computational methods in different aspects of transporters research. In the absence of a high-resolution structure of most of transporters, homology modeling is a useful tool to interpret experimental data and potentially guide experimental studies. We summarize reported homology modeling in this review. Researches in computational methods cover major members of transporters and a variety of topics including the classification of substrates and/or inhibitors, prediction of protein-ligand interactions, constitution of binding pocket, phenotype of non-synonymous single-nucleotide polymorphisms, and the conformation analysis that try to explain the mechanism of action. As an example, one of the most important transporters P-gp is elaborated to explain the differences and advantages of various computational models. In the third part, the challenges of developing computational methods to get reliable prediction, as well as the potential future directions in transporter related modeling are discussed.

  1. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    PubMed

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

  2. HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

    PubMed Central

    Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

    2010-01-01

    Background Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death. PMID:20186341

  3. Fluorescence interference contrast based approach to study real time interaction of melittin with plasma membranes

    NASA Astrophysics Data System (ADS)

    Gupta, Sharad; Gui, Dong; Zandi, Roya; Gill, Sarjeet; Mohideen, Umar

    2014-03-01

    Melittin is an anti-bacterial and hemolytic toxic peptide found in bee venom. Cell lysis behavior of peptides has been widely investigated, but the exact interaction mechanism of lytic peptides with lipid membranes and its constituents has not been understood completely. In this paper we study the melittin interaction with lipid plasma membranes in real time using non-invasive and non-contact fluorescence interference contrast microscopy (FLIC). Particularly the interaction of melittin with plasma membranes was studied in a controlled molecular environment, where these plasma membrane were composed of saturated lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and unsaturated lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) with and without cholesterol. We found out that melittin starts to form nanometer size pores in the plasma membranes shortly after interacting with membranes. But the addition of cholesterol in plasma membrane slows down the pore formation process. Our results show that inclusion of cholesterol to the plasma membranes make them more resilient towards pore formation and lysis of membrane.

  4. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    PubMed Central

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  5. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  6. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  7. Interaction between plant polyphenols and the erythrocyte membrane.

    PubMed

    Cyboran, Sylwia; Oszmiański, Jan; Kleszczyńska, Halina

    2012-03-01

    The purpose of these studies was to determine the effect of polyphenols contained in extracts from apple, strawberry and blackcurrant on the properties of the erythrocyte membrane, treated as a model of the biological membrane. To this end, the effect of the substances used on hemolysis, osmotic resistance and shape of erythrocytes, and on packing order in the hydrophilic region of the erythrocyte membrane was studied. The investigation was performed with spectrophotometric and fluorimetric methods, and using the optical microscope. The hemolytic studies have shown that the extracts do not induce hemolysis at the concentrations used. The results obtained from the spectrophotometric measurements of osmotic resistance of erythrocytes showed that the polyphenols contained in the extracts cause an increase in the resistance, rendering them less prone to hemolysis in hypotonic solutions of sodium chloride. The fluorimetric studies indicate that the used substances cause a decrease of packing order in the hydrophilic area of membrane lipids. The observations of erythrocyte shapes in a biological optical microscope have shown that, as a result of the substances' action, the erythrocytes become mostly echinocytes, which means that the polyphenols of the extracts localize in the outer lipid monolayer of the erythrocyte membrane. The results obtained indicate that, in the concentration range used, the plant extracts are incorporated into the hydrophilic area of the membrane, modifying its properties.

  8. Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody.

    PubMed

    Yu, Deqiang; Chen, Xiaonong; Pelton, Robert; Ghosh, Raja

    2008-04-15

    This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.

  9. Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane

    PubMed Central

    Wenzel, Michaela; Kohl, Bastian; Münch, Daniela; Raatschen, Nadja; Albada, H. Bauke; Hamoen, Leendert; Metzler-Nolte, Nils; Sahl, Hans-Georg

    2012-01-01

    Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane. PMID:22926563

  10. Monte Carlo simulations of peptide-membrane interactions with the MCPep web server.

    PubMed

    Gofman, Yana; Haliloglu, Turkan; Ben-Tal, Nir

    2012-07-01

    The MCPep server (http://bental.tau.ac.il/MCPep/) is designed for non-experts wishing to perform Monte Carlo (MC) simulations of helical peptides in association with lipid membranes. MCPep is a web implementation of a previously developed MC simulation model. The model has been tested on a variety of peptides and protein fragments. The simulations successfully reproduced available empirical data and provided new molecular insights, such as the preferred locations of peptides in the membrane and the contribution of individual amino acids to membrane association. MCPep simulates the peptide in the aqueous phase and membrane environments, both described implicitly. In the former, the peptide is subjected solely to internal conformational changes, and in the latter, each MC cycle includes additional external rigid body rotational and translational motions to allow the peptide to change its location in the membrane. The server can explore the interaction of helical peptides of any amino-acid composition with membranes of various lipid compositions. Given the peptide's sequence or structure and the natural width and surface charge of the membrane, MCPep reports the main determinants of peptide-membrane interactions, e.g. average location and orientation in the membrane, free energy of membrane association and the peptide's helical content. Snapshots of example simulations are also provided.

  11. Membrane interaction of the factor VIIIa discoidin domains in atomistic detail

    PubMed Central

    Madsen, Jesper J.; Ohkubo, Y. Zenmei; Peters, Günther H.; Faber, Johan H.; Tajkhorshid, Emad; Olsen, Ole H.

    2016-01-01

    A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor (F)VIIIa, the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla)-domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent non-specificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2. PMID:26346528

  12. Preferential Affirmative Action.

    ERIC Educational Resources Information Center

    Bell, Derrick A., Jr.

    1982-01-01

    Discusses the philosophical rationale for preferential affirmative action presented by Daniel C. Maguire in "A New American Justice." Maintains that self-interest bars present society's acceptance of Maguire's theories of justice, as demonstrated in negative reactions to the Harvard Law Review's affirmative action plan. (MJL)

  13. Against Preferential Treatment.

    ERIC Educational Resources Information Center

    Kekes, John

    1997-01-01

    Argues that preferential treatment of women and minorities in the selection of college faculty elevates a form of corruption to standard administrative practice by including people in academic life on the basis of characteristics irrelevant to teaching and research; and previous unjust treatment is inadequate justification for preferential…

  14. Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

    PubMed

    Wenk, M R; Seelig, J

    1997-11-01

    Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa

  15. Kinetics of endophilin N-BAR domain dimerization and membrane interactions.

    PubMed

    Capraro, Benjamin R; Shi, Zheng; Wu, Tingting; Chen, Zhiming; Dunn, Joanna M; Rhoades, Elizabeth; Baumgart, Tobias

    2013-05-01

    The recruitment to plasma membrane invaginations of the protein endophilin is a temporally regulated step in clathrin-mediated endocytosis. Endophilin is believed to sense or stabilize membrane curvature, which in turn likely depends on the dimeric structure of the protein. The dynamic nature of the membrane association and dimerization of endophilin is thus functionally important and is illuminated herein. Using subunit exchange Förster resonance energy transfer (FRET), we determine dimer dissociation kinetics and find a dimerization equilibrium constant orders of magnitude lower than previously published values. We characterize N-BAR domain membrane association kinetics under conditions where the dimeric species predominates, by stopped flow, observing prominent electrostatic sensitivity of membrane interaction kinetics. Relative to membrane binding, we find that protein monomer/dimer species equilibrate with far slower kinetics. Complementary optical microscopy studies reveal strikingly slow membrane dissociation and an increase of dissociation rate constant for a construct lacking the amphipathic segment helix 0 (H0). We attribute the slow dissociation kinetics to higher-order protein oligomerization on the membrane. We incorporate our findings into a kinetic scheme for endophilin N-BAR membrane binding and find a significant separation of time scales for endophilin membrane binding and subsequent oligomerization. This separation may facilitate the regulation of membrane trafficking phenomena.

  16. Radiation Interaction with Therapeutic Drugs and Cell Membranes

    NASA Astrophysics Data System (ADS)

    Martin, Diana I.; Manaila, Elena N.; Moisescu, Mihaela I.; Savopol, Tudor D.; Kovacs, Eugenia A.; Cinca, Sabin A.; Matei, Constantin I.; Margaritescu, Irina D.; Iacob, Nicusor I.; Ighigeanu, Daniel I.; Craciun, Gabriela D.

    2007-04-01

    This transient permeabilized state of the cell membrane, named the ``cell electroporation'' (CE) can be used to increase cells uptake of drugs that do not readily pass cell membrane, thus enabling their cytotoxicity. The anticancer drugs, such as bleomycin (BL) and cisplatin, are the most candidates for the combined use with ionizing and non-ionizing radiation fields. The methods and installations for the cell electroporation by electron beam (EB) and microwave (MW) irradiation are presented. The viability tests of the human leukocytes under EB and MW exposure with/without the BL in the cell cultures are discussed.

  17. Radiation Interaction with Therapeutic Drugs and Cell Membranes

    SciTech Connect

    Martin, Diana I.; Manaila, Elena N.; Matei, Constantin I.; Iacob, Nicusor I.; Ighigeanu, Daniel I.; Craciun, Gabriela D.; Moisescu, Mihaela I.; Savopol, Tudor D.; Kovacs, Eugenia A.; Cinca, Sabin A.; Margaritescu, Irina D.

    2007-04-23

    This transient permeabilized state of the cell membrane, named the 'cell electroporation' (CE) can be used to increase cells uptake of drugs that do not readily pass cell membrane, thus enabling their cytotoxicity. The anticancer drugs, such as bleomycin (BL) and cisplatin, are the most candidates for the combined use with ionizing and non-ionizing radiation fields. The methods and installations for the cell electroporation by electron beam (EB) and microwave (MW) irradiation are presented. The viability tests of the human leukocytes under EB and MW exposure with/without the BL in the cell cultures are discussed.

  18. Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B.

    PubMed

    Cordeiro, André M; Figueiredo, Duarte D; Tepperman, James; Borba, Ana Rita; Lourenço, Tiago; Abreu, Isabel A; Ouwerkerk, Pieter B F; Quail, Peter H; Margarida Oliveira, M; Saibo, Nelson J M

    2016-02-01

    DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses. PMID:26732823

  19. Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of Phytochrome B

    PubMed Central

    Cordeiro, André M.; Figueiredo, Duarte D.; Tepperman, James; Borba, Ana Rita; Lourenço, Tiago; Abreu, Isabel A.; Ouwerkerk, Pieter B.F.; Quail, Peter H.; Oliveira, M. Margarida; Saibo, Nelson J. M.

    2016-01-01

    DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a Yeast one Hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a Phytochrome Interacting Factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses. PMID:26732823

  20. Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion.

    PubMed

    He, Tao; Gershenson, Anne; Eyles, Stephen J; Lee, Yan-Jiun; Liu, Wenshe R; Wang, Jiangyun; Gao, Jianmin; Roberts, Mary F

    2015-07-31

    Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins. PMID:26092728

  1. Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion*

    PubMed Central

    He, Tao; Gershenson, Anne; Eyles, Stephen J.; Lee, Yan-Jiun; Liu, Wenshe R.; Wang, Jiangyun; Gao, Jianmin; Roberts, Mary F.

    2015-01-01

    Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins. PMID:26092728

  2. Studies on the interactions of bisphenols with anionic phospholipids of decomposer membranes in model systems.

    PubMed

    Broniatowski, Marcin; Sobolewska, Katarzyna; Flasiński, Michał; Wydro, Paweł

    2016-04-01

    Bisphenol A (BPA) and other bisphenols constitute a class of organic pollutants, which because of their estrogenic properties, low dose activity and bioaccumulation pose considerable risk for public health as well as for the environment. Accumulated in the sediment bisphenols can endanger the decomposers' populations being incorporated into their cellular membranes; however, the mechanism of their membrane activity is unknown. Therefore, to study these phenomena we applied anionic phospholipid Langmuir monolayers as simple but versatile models of decomposers biomembranes. Phosphatidylglycerols and cardiolipins are not only the main components of bacterial membranes but also of crucial importance in mitochondrial and thylakoid membranes in eukaryotic cells. In our investigations we applied five compounds of the bisphenol class most commonly detected in the environment. To characterize the bisphenols-model membrane interactions we applied multiple mutually independent methods of physical chemistry; namely: the Langmuir monolayer technique, surface potential measurements, Brewster angle microscopy for the visualization of the monolayers' texture and grazing incidence X-ray diffraction for the discussion of the phospholipids packing within the monolayers. Our studies indicated that all the investigated bisphenols interact with the model membrane, but the strength of the interactions is dependent on the bisphenol structure and hydrophobicity and the fluidity of the model membranes. We proved that bisphenol S often treated as the least toxic BPA analog can also be incorporated to the model membranes changing their structure and fluidity. PMID:26806160

  3. Interaction between La(III) and proteins on the plasma membrane of horseradish

    NASA Astrophysics Data System (ADS)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  4. Studies on the interactions of bisphenols with anionic phospholipids of decomposer membranes in model systems.

    PubMed

    Broniatowski, Marcin; Sobolewska, Katarzyna; Flasiński, Michał; Wydro, Paweł

    2016-04-01

    Bisphenol A (BPA) and other bisphenols constitute a class of organic pollutants, which because of their estrogenic properties, low dose activity and bioaccumulation pose considerable risk for public health as well as for the environment. Accumulated in the sediment bisphenols can endanger the decomposers' populations being incorporated into their cellular membranes; however, the mechanism of their membrane activity is unknown. Therefore, to study these phenomena we applied anionic phospholipid Langmuir monolayers as simple but versatile models of decomposers biomembranes. Phosphatidylglycerols and cardiolipins are not only the main components of bacterial membranes but also of crucial importance in mitochondrial and thylakoid membranes in eukaryotic cells. In our investigations we applied five compounds of the bisphenol class most commonly detected in the environment. To characterize the bisphenols-model membrane interactions we applied multiple mutually independent methods of physical chemistry; namely: the Langmuir monolayer technique, surface potential measurements, Brewster angle microscopy for the visualization of the monolayers' texture and grazing incidence X-ray diffraction for the discussion of the phospholipids packing within the monolayers. Our studies indicated that all the investigated bisphenols interact with the model membrane, but the strength of the interactions is dependent on the bisphenol structure and hydrophobicity and the fluidity of the model membranes. We proved that bisphenol S often treated as the least toxic BPA analog can also be incorporated to the model membranes changing their structure and fluidity.

  5. Interaction of the NMDA receptor noncompetitive antagonist MK-801 with model and native membranes.

    PubMed Central

    Moring, J; Niego, L A; Ganley, L M; Trumbore, M W; Herbette, L G

    1994-01-01

    MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo. PMID:7696477

  6. Interaction of Lamb modes with two-level systems in amorphous nanoscopic membranes.

    SciTech Connect

    Kuhn, T.; Anghel, D. V.; Galperin, Y. M.; Manninen, M.; Materials Science Division; Univ. Jyvaskyla; National Inst. for Physics and Nuclear Engineering; Bogolivbov Lab. Theoretical Physics; Univ. Oslo; Russian Academy of Sciences

    2007-01-01

    Using a generalized model of interaction between a two-level system (TLS) and an arbitrary deformation of the material, we calculate the interaction of Lamb modes with TLSs in amorphous nanoscopic membranes. We compare the mean free paths of the Lamb modes of different symmetries and calculate the heat conductivity {kappa}. In the limit of an infinitely wide membrane, the heat conductivity is divergent. Nevertheless, the finite size of the membrane imposes a lower cutoff for the phonon frequencies, which leads to the temperature dependence {kappa}{alpha}T(a+b ln T). This temperature dependence is a hallmark of the TLS-limited heat conductance at low temperature.

  7. Membrane recognition by vesicular stomatitis virus involves enthalpy-driven protein-lipid interactions.

    PubMed

    Carneiro, Fabiana A; Bianconi, M Lucia; Weissmüller, Gilberto; Stauffer, Fausto; Da Poian, Andrea T

    2002-04-01

    Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.

  8. Membrane Recognition by Vesicular Stomatitis Virus Involves Enthalpy-Driven Protein-Lipid Interactions

    PubMed Central

    Carneiro, Fabiana A.; Bianconi, M. Lucia; Weissmüller, Gilberto; Stauffer, Fausto; Da Poian, Andrea T.

    2002-01-01

    Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process. PMID:11907215

  9. What does make an amyloid toxic: morphology, structure or interaction with membrane?

    PubMed

    Berthelot, Karine; Cullin, Christophe; Lecomte, Sophie

    2013-01-01

    The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a "raft-like" mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.

  10. Measuring Selective Estrogen Receptor Modulator (SERM)–Membrane Interactions with Second Harmonic Generation

    PubMed Central

    2015-01-01

    The interaction of selective estrogen receptor modulators (SERMs) with lipid membranes has been measured at clinically relevant serum concentrations using the label-free technique of second harmonic generation (SHG). The SERMs investigated in this study include raloxifene, tamoxifen, and the tamoxifen metabolites 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. Equilibrium association constants (Ka) were measured for SERMs using varying lipid compositions to examine how lipid phase, packing density, and cholesterol content impact SERM-membrane interactions. Membrane-binding properties of tamoxifen and its metabolites were compared on the basis of hydroxyl group substitution and amine ionization to elucidate how the degree of drug ionization impacts membrane partitioning. SERM-membrane interactions were probed under multiple pH conditions, and drug adsorption was observed to vary with the concentration of soluble neutral species. The agreement between Ka values derived from SHG measurements of the interactions between SERMs and artificial cell membranes and independent observations of the SERMs efficacy from clinical studies suggests that quantifying membrane adsorption properties may be important for understanding SERM action in vivo. PMID:24410282

  11. Interaction of C60 fullerenes with asymmetric and curved lipid membranes: a molecular dynamics study.

    PubMed

    Cherniavskyi, Yevhen K; Ramseyer, Christophe; Yesylevskyy, Semen O

    2016-01-01

    Interaction of fullerenes with asymmetric and curved DOPC/DOPS bicelles is studied by means of coarse-grained molecular dynamics simulations. The effects caused by asymmetric lipid composition of the membrane leaflets and the curvature of the membrane are analyzed. It is shown that the aggregates of fullerenes prefer to penetrate into the membrane in the regions of the moderately positive mean curvature. Upon penetration into the hydrophobic core of the membrane fullerenes avoid the regions of the extreme positive or the negative curvature. Fullerenes increase the ordering of lipid tails, which are in direct contact with them, but do not influence other lipids significantly. Our data suggest that the effects of the membrane curvature should be taken into account in the studies concerning permeability of the membranes to fullerenes and fullerene-based drug delivery systems.

  12. Electrostatic interaction effects on tension-induced pore formation in lipid membranes

    NASA Astrophysics Data System (ADS)

    Karal, Mohammad Abu Sayem; Levadnyy, Victor; Tsuboi, Taka-aki; Belaya, Marina; Yamazaki, Masahito

    2015-07-01

    We investigated the effects of electrostatic interactions on the rate constant (kp) for tension-induced pore formation in lipid membranes of giant unilamellar vesicles under constant applied tension. A decrease in salt concentration in solution as well as an increase in surface charge density of the membranes increased kp. These data indicate that kp increases as the extent of electrostatic interaction increases. We developed a theory on the effect of the electrostatic interactions on the free energy profile of the membrane containing a prepore and also on the values of kp; this theory explains the experimental results and fits the experimental data reasonably well in the presence of weak electrostatic interactions. Based on these results, we conclude that a decrease in the free energy barrier of the prepore state due to electrostatic interactions is the main factor causing an increase in kp.

  13. Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

    PubMed

    Broecker, Jana; Fiedler, Sebastian; Gimpl, Katharina; Keller, Sandro

    2014-10-01

    Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.

  14. Controlled CO preferential oxidation

    DOEpatents

    Meltser, Mark A.; Hoch, Martin M.

    1997-01-01

    Method for controlling the supply of air to a PROX reactor for the preferential oxidation in the presence of hydrogen wherein the concentration of the hydrogen entering and exiting the PROX reactor is monitored, the difference therebetween correlated to the amount of air needed to minimize such difference, and based thereon the air supply to the PROX reactor adjusted to provide such amount and minimize such difference.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  16. Interaction of Serum Proteins with Surface of Hemodialysis Fiber Membranes

    NASA Astrophysics Data System (ADS)

    Afrin, Rehana; Shirako, Yuji; Kishimoto, Kikuo; Ikai, Atsushi

    2012-08-01

    The poly(vinyl pyrrolidone)-covered hydrophilic surface of hollow-fiber membranes (fiber membrane, hereafter) for hemodialysis was mechanically probed using modified tips on an atomic force microscope (AFM) with covalent crosslinkers and several types of serum protein. The retraction part of many of the force extension (F-E) curves obtained with AFM tips coated with serum albumin had a long and smooth extension up to 200-300 nm indicating forced elongation of poly(vinyl pyrrolidone) chains. When fibrinogen-coated tips were used, long extension F-E curves up to 500 nm with multiple peaks were obtained in addition to smooth curves most likely reflecting the unfolding of fibrinogen molecules. The results indicated that individual polymer chains had a significant affinity toward serum proteins. The adhesion frequency of tips coated with serum proteins was lower on the poly(vinyl pyrrolidone) surface than on the uncoated hydrophobic polysulfone surface.

  17. Polymeric blend nanocomposite membranes for ethanol dehydration-effect of morphology and membrane-solvent interactions

    EPA Science Inventory

    Nanocomposite membranes (NCMs) of sodium alginate/poly(vinyl pyrrolidone) blend polymers incorporated with varying concentrations of phosphotungstic acid (H3PW12O40) (PWA) nanoparticles have been prepared and used in ethanol dehydration by the pervaporation (PV) technique. Effe...

  18. Axon-glia interaction and membrane traffic in myelin formation

    PubMed Central

    White, Robin; Krämer-Albers, Eva-Maria

    2014-01-01

    In vertebrate nervous systems myelination of neuronal axons has evolved to increase conduction velocity of electrical impulses with minimal space and energy requirements. Myelin is formed by specialized glial cells which ensheath axons with a lipid-rich insulating membrane. Myelination is a multi-step process initiated by axon-glia recognition triggering glial polarization followed by targeted myelin membrane expansion and compaction. Thereby, a myelin sheath of complex subdomain structure is established. Continuous communication between neurons and glial cells is essential for myelin maintenance and axonal integrity. A diverse group of diseases, from multiple sclerosis to schizophrenia, have been linked to malfunction of myelinating cells reflecting the physiological importance of the axon-glial unit. This review describes the mechanisms of axonal signal integration by oligodendrocytes emphasizing the central role of the Src-family kinase Fyn during central nervous system (CNS) myelination. Furthermore, we discuss myelin membrane trafficking with particular focus on endocytic recycling and the control of proteolipid protein (PLP) transport by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Finally, PLP mistrafficking is considered in the context of myelin diseases. PMID:24431989

  19. Interaction of general anesthetics with phospholipid vesicles and biological membranes.

    PubMed

    Vanderkooi, J M; Landesberg, R; Selick, H; McDonald, G G

    1977-01-01

    Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6-phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.

  20. Assessing the effect of surface modification of polyamide RO membrane by l-DOPA on the short range physiochemical interactions with biopolymer fouling on the membrane.

    PubMed

    Azari, Sara; Zou, Linda; Cornelissen, Emile

    2014-08-01

    Theoretical predictions of interaction energies for membrane-biopolymer foulant pairs were used to compare the fouling tendencies of a virgin commercial polyamide reverse osmosis (RO) membrane with a amino acid 3-(3,4-dihydroxyphenyl)-l-alanine (l-DOPA) coated RO membrane. Lifshitz-van der Waals (LW) and Lewis acid-base (AB) surface tension components of the membranes were determined based on contact angle results using the van Oss approach. From these values, the LW and AB components of the free energy of adhesion between membrane and foulants were calculated. Electrostatic (EL) double layer interaction energies between the membrane and foulants were also estimated using the measured surface charge data of the membranes and fouling agents. Bovine serum albumin (BSA) and alginic acid sodium salt (alginate) were used as model biopolymers causing membrane fouling. Based on the calculated adhesion free energies, acid-base interactions were found to have the strongest impact on the adhesion of both BSA and alginate to the either membranes surfaces. It was found that l-DOPA modification has significantly lowered acid-base interaction affinity toward the adhesion of both foulants studied. On the basis of calculated free energies of adhesion, lower fouling tendency of the l-DOPA modified membrane was expected. The accelerated fouling tests indicated a lower flux decline rate for the modified membrane and confirmed the results obtained from theory.

  1. Liquid crystals and their interactions with colloidal particles and phospholipid membranes: Molecular simulation studies

    NASA Astrophysics Data System (ADS)

    Kim, Evelina B.

    Experimentally, liquid crystals (LC) can be used as the basis for optical biomolecular sensors that rely on LC ordering. Recently, the use of LC as a reporting medium has been extended to investigations of molecular scale processes at lipid laden aqueous-LC interfaces and at biological cell membranes. In this thesis, we present two related studies where liquid crystals are modelled at different length scales. We examine (a) the behavior of nanoscopic colloidal particles in LC systems, using Monte Carlo (MC) molecular simulations and a mesoscopic dynamic field theory (DyFT); and (b) specific interactions of two types of mesogens with a model phospholipid bilayer, using atomistic molecular dynamics (MD) at the A-nm scale. In (a), we consider colloidal particles suspended in a LC, confined between two walls. We calculate the colloid-substrate and colloid-colloid potentials of mean force (PMF). For the MC simulations, we developed a new technique (ExEDOS or Expanded Ensemble Density Of States) that ensures good sampling of phase space without prior knowledge of the energy landscape of the system. Both results, simulation and DyFT, indicate a repulsive force acting between a colloid and a wall. In contrast, both techniques indicate an overall colloid-colloid attraction and predict a new topology of the disclination lines that arises when the particles approach each other. In (b), we find that mesogens (pentylcyanobiphenyl [5CB] or difluorophenyl-pentylbicyclohexyl [5CF]) preferentially partition from the aqueous phase into a dipalmitoylphosphatidylcholine (DPPC) bilayer. We find highly favorable free energy differences for partitioning (-18kBT for 5CB, -26k BT for 5CF). We also simulated fully hydrated bilayers with embedded 5CB or 5CF at concentrations used in recent experiments (6 mol% and 20 mol%). The presence of mesogens in the bilayer enhances the order of lipid acyl tails and changes the spatial and orientational arrangement of lipid headgroup atoms. A stronger

  2. The role of hydrophobic interactions in positioning of peripheral proteins in membranes

    PubMed Central

    Lomize, Andrei L; Pogozheva, Irina D; Lomize, Mikhail A; Mosberg, Henry I

    2007-01-01

    Background Three-dimensional (3D) structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined. Results We report the first large-scale computational study of monotopic/peripheral proteins with known 3D structures. The optimal translational and rotational positions of 476 proteins are determined by minimizing energy of protein transfer from water to the lipid bilayer, which is approximated by a hydrocarbon slab with a decadiene-like polarity and interfacial regions characterized by water-permeation profiles. Predicted membrane-binding sites, protein tilt angles and membrane penetration depths are consistent with spin-labeling, chemical modification, fluorescence, NMR, mutagenesis, and other experimental studies of 53 peripheral proteins and peptides. Experimental membrane binding affinities of peripheral proteins were reproduced in cases that did not involve a helix-coil transition, specific binding of lipids, or a predominantly electrostatic association. Coordinates of all examined peripheral proteins and peptides with the calculated hydrophobic membrane boundaries, subcellular localization, topology, structural classification, and experimental references are available through the Orientations of Proteins in Membranes (OPM) database. Conclusion Positions of diverse peripheral proteins and peptides in the lipid bilayer can be accurately predicted using their 3D structures that represent a proper membrane-bound conformation and oligomeric state, and have membrane binding elements present. The success of the implicit solvation model suggests that hydrophobic interactions are usually sufficient to determine the spatial position of a protein in the membrane, even when electrostatic interactions or specific binding of lipids are substantial. Our results demonstrate that

  3. Study of interactions between polymer nanoparticles and cell membranes at atomistic levels.

    PubMed

    Yong, Chin W

    2015-02-01

    Knowledge of how the structure of nanoparticles and the interactions with biological cell membranes is important not only for understanding nanotoxicological effects on human, animal health and the environment, but also for better understanding of nanoparticle fabrication for biomedical applications. In this work, we use molecular modelling techniques, namely molecular dynamics (MD) simulations, to explore how polymer nanoparticles interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid cell membranes. Two different polymers have been considered: 100 monomer units of polyethylene (approx. 2.83 kDa) and polystyrene (approx. 10.4 kDa), both of which have wide industrial applications. We found that, despite the polar lipid head groups acting as an effective barrier to prevent the nanoparticles from interacting with the membrane surface, irreversible adhesion can be initiated by insertion of dangling chain ends from the polymer into the hydrophobic interior of the membrane. In addition, alignment of chain segments from the polymers with that of hydrocarbon chains in the interior of the membrane facilitates the complete immersion of the nanoparticles into the cell membrane. These findings highlight the importance of the surface and the topological structures of the polymer particles that dictate the absorption behaviour into the membrane and, subsequently, induce the possible translocation into the cell.

  4. Bacteria-polymeric membrane interactions: atomic force microscopy and XDLVO predictions.

    PubMed

    Thwala, Justice M; Li, Minghua; Wong, Mavis C Y; Kang, Seoktae; Hoek, Eric M V; Mamba, Bhekie B

    2013-11-12

    Atomic force microscopy (AFM) in conjunction with a bioprobe developed using a polydopamine wet adhesive was used to directly measure the adhesive force between bacteria and different polymeric membrane surfaces. Bacterial cells of Pseudomonas putida and Bacillus subtilis were immobilized onto the tip of a standard AFM cantilever, and force measurements made using the modified cantilever on various membranes. Interaction forces measured with the bacterial probe were compared, qualitatively, to predictions by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory with steric interactions included. The XDLVO theory predicted attractive interactions between low energy hydrophobic membranes with high energy hydrophilic bacterium (P. putida). It also predicted a shallow primary maximum with the most hydrophilic bacterium, B. subtilis . Discrepancies between predictions using the XDLVO theory and theory require involvement of factors such as bridging effects. Differences in interaction between P. putida and B. subtilis are attributed to acid-base interactions and steric interactions. P. putida is Gram negative with lipopolysaccharides present in the outer cell membrane. A variation in forces of adhesion for bacteria on polymeric membranes studied was interpreted in terms of hydrophilicity and interfacial surface potential calculated from physicochemical properties.

  5. Molecular Dynamics Simulations of Amyloid β-Peptide (1-42): Tetramer Formation and Membrane Interactions.

    PubMed

    Brown, Anne M; Bevan, David R

    2016-09-01

    The aggregation cascade and peptide-membrane interactions of the amyloid β-peptide (Aβ) have been implicated as toxic events in the development and progression of Alzheimer's disease. Aβ42 forms oligomers and ultimately plaques, and it has been hypothesized that these oligomeric species are the main toxic species contributing to neuronal cell death. To better understand oligomerization events and subsequent oligomer-membrane interactions of Aβ42, we performed atomistic molecular-dynamics (MD) simulations to characterize both interpeptide interactions and perturbation of model membranes by the peptides. MD simulations were utilized to first show the formation of a tetramer unit by four separate Aβ42 peptides. Aβ42 tetramers adopted an oblate ellipsoid shape and showed a significant increase in β-strand formation in the final tetramer unit relative to the monomers, indicative of on-pathway events for fibril formation. The Aβ42 tetramer unit that formed in the initial simulations was used in subsequent MD simulations in the presence of a pure POPC or cholesterol-rich raft model membrane. Tetramer-membrane simulations resulted in elongation of the tetramer in the presence of both model membranes, with tetramer-raft interactions giving rise to the rearrangement of key hydrophobic regions in the tetramer and the formation of a more rod-like structure indicative of a fibril-seeding aggregate. Membrane perturbation by the tetramer was manifested in the form of more ordered, rigid membranes, with the pure POPC being affected to a greater extent than the raft membrane. These results provide critical atomistic insight into the aggregation pathway of Aβ42 and a putative toxic mechanism in the pathogenesis of Alzheimer's disease. PMID:27602722

  6. Interaction of Spin-Labeled Lipid Membranes with Transition Metal Ions

    PubMed Central

    2015-01-01

    The large values of spin relaxation enhancement (RE) for PC spin-labels in the phospholipid membrane induced by paramagnetic metal salts dissolved in the aqueous phase can be explained by Heisenberg spin exchange due to conformational fluctuations of the nitroxide group as a result of membrane fluidity, flexibility of lipid chains, and, possibly, amphiphilic nature of the nitroxide label. Whether the magnetic interaction occurs predominantly via Heisenberg spin exchange (Ni) or by the dipole–dipole (Gd) mechanism, it is essential for the paramagnetic ion to get into close proximity to the nitroxide moiety for efficient RE. For different salts of Ni the RE in phosphatidylcholine membranes follows the anionic Hofmeister series and reflects anion adsorption followed by anion-driven attraction of paramagnetic cations on the choline groups. This adsorption is higher for chaotropic ions, e.g., perchlorate. (A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules.) However, there is no anionic dependence of RE for model membranes made from negatively charged lipids devoid of choline groups. We used Ni-induced RE to study the thermodynamics and electrostatics of ion/membrane interactions. We also studied the effect of membrane composition and the phase state on the RE values. In membranes with cholesterol a significant difference is observed between PC labels with nitroxide tethers long enough vs not long enough to reach deep into the membrane hydrophobic core behind the area of fused cholesterol rings. This study indicates one must be cautious in interpreting data obtained by PC labels in fluid membranes in terms of probing membrane properties at different immersion depths when it can be affected by paramagnetic species at the membrane surface. PMID:26490692

  7. Inter-helical interactions in membrane proteins: analysis based on the local backbone geometry and the side chain interactions.

    PubMed

    Jha, Anupam Nath; Vishveshwara, Saraswathi

    2009-06-01

    The availability of a significant number of the structures of helical membrane proteins has prompted us to investigate the mode of helix-helix packing. In the present study, we have considered a dataset of alpha-helical membrane proteins representing structures solved from all the known superfamilies. We have described the geometry of all the helical residues in terms of local coordinate axis at the backbone level. Significant inter-helical interactions have been considered as contacts by weighing the number of atom-atom contacts, including all the side-chain atoms. Such a definition of local axis and the contact criterion has allowed us to investigate the inter-helical interaction in a systematic and quantitative manner. We show that a single parameter (designated as alpha), which is derived from the parameters representing the mutual orientation of local axes, is able to accurately capture the details of helix-helix interaction. The analysis has been carried out by dividing the dataset into parallel, anti-parallel, and perpendicular orientation of helices. The study indicates that a specific range of alpha value is preferred for interactions among the anti-parallel helices. Such a preference is also seen among interacting residues of parallel helices, however to a lesser extent. No such preference is seen in the case of perpendicular helices, the contacts that arise mainly due to the interaction of surface helices with the end of the trans-membrane helices. The study supports the prevailing view that the anti-parallel helices are well packed. However, the interactions between helices of parallel orientation are non-trivial. The packing in alpha-helical membrane proteins, which is systematically and rigorously investigated in this study, may prove to be useful in modeling of helical membrane proteins.

  8. Prediction of the most favorable configuration in the ACBP-membrane interaction based on electrostatic calculations.

    PubMed

    Vallejo, Diego F; Zamarreño, Fernando; Guérin, Diego M A; Grigera, J Raul; Costabel, Marcelo D

    2009-03-01

    Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.

  9. Assessment of sulfur mustard interaction with basement membrane components

    SciTech Connect

    Zhang, Z.; Peters, B.P.; Monteiro-Rivier, N.A.

    1995-08-01

    Bis-2-chloroethyl sulfide (sulfur mustard, RD) is a bifunctional alkylating agent which causes severe vesication characterized by slow wound healing. Our previous studies have shown that the vesicant RD disrupts the epidermal-dermal junction at the lamina lucida of the basement membrane. The purpose of this study was to examine whether RD directly modifies basement membrane components (BMCs), and to evaluate the effect of RD on the cell adhesive activity of BMCs. EHS laminin was incubated with (14C)HRD, and extracted by gel filtration. Analysis of the (14C)HRD-conjugated laminin fraction by a reduced sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SD S-PAGE) revealed the incorporation of radioactivity into both laminin subunits and a laminin trimer resistant to dissociation in reduced SDS-PAGE sample buffer, suggesting direct alkylation and cross-linking of EHS laminin by (14C)HD. Normal human foreskin epidermal keratinocytes were biosynthetically labeled with (35S)cysteine. (35S)-labeled laminin isoforms, Ae.Ble.B2e. laminin and K.Ble.B2e. laminin (using the nomenclature of Engel), fibronectin, and heparan sulfate proteoglycan were isolated by irnmunoprecipitation from the cell culture medium, treated with RD or ethanol as control, and then analyzed by SDS-PAGE.

  10. Mapping membrane protein interactions in cell signaling systems.

    SciTech Connect

    Light, Yooli Kim; Hadi, Masood Z.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J.; Wood, Nichole L.; Schoeniger, Joseph S.; Young, Malin M.

    2003-12-01

    We proposed to apply a chemical cross-linking, mass spectrometry and modeling method called MS3D to the structure determination of the rhodopsin-transducin membrane protein complex (RTC). Herein we describe experimental progress made to adapt the MS3D approach for characterizing membrane protein systems, and computational progress in experimental design, data analysis and protein structure modeling. Over the past three years, we have developed tailored experimental methods for all steps in the MS3D method for rhodopsin, including protein purification, a functional assay, cross-linking, proteolysis and mass spectrometry. In support of the experimental effort. we have out a data analysis pipeline in place that automatically selects the monoisotopic peaks in a mass spectrometric spectrum, assigns them and stores the results in a database. Theoretical calculations using 24 experimentally-derived distance constraints have resulted in a backbone-level model of the activated form of rhodopsin, which is a critical first step towards building a model of the RTC. Cross-linked rhodopsin-transducin complexes have been isolated via gel electrophoresis and further mass spectrometric characterization of the cross-links is underway.

  11. Cooperative Gating and Spatial Organization of Membrane Proteins through Elastic Interactions

    PubMed Central

    Ursell, Tristan; Huang, Kerwyn Casey; Peterson, Eric; Phillips, Rob

    2007-01-01

    Biological membranes are elastic media in which the presence of a transmembrane protein leads to local bilayer deformation. The energetics of deformation allow two membrane proteins in close proximity to influence each other's equilibrium conformation via their local deformations, and spatially organize the proteins based on their geometry. We use the mechanosensitive channel of large conductance (MscL) as a case study to examine the implications of bilayer-mediated elastic interactions on protein conformational statistics and clustering. The deformations around MscL cost energy on the order of 10 kBT and extend ∼3 nm from the protein edge, as such elastic forces induce cooperative gating, and we propose experiments to measure these effects. Additionally, since elastic interactions are coupled to protein conformation, we find that conformational changes can severely alter the average separation between two proteins. This has important implications for how conformational changes organize membrane proteins into functional groups within membranes. PMID:17480116

  12. Interaction of SMKT, a killer toxin produced by Pichia farinosa, with the yeast cell membranes.

    PubMed

    Suzuki, C; Ando, Y; Machida, S

    2001-12-01

    SMKT (salt-mediated killer toxin), a killer toxin produced by the halotolerant yeast, Pichia farinosa, kills yeasts of several genera, including Saccharomyces cerevisiae. To elucidate the killing mechanism of SMKT, we examined the interaction of SMKT with membranes using liposomes. Leakage of calcein from calcein-entrapped liposomes was observed in the presence of SMKT. Destruction of liposomes was observed by dark-field microscopy. Comparison of intact S. cerevisiae cells with SMKT-treated cells by dark-field microscopy indicated that the spherical cell membrane is disrupted by SMKT. Using sodium carbonate extraction, we obtained direct evidence for the first time that SMKT is associated with the membrane of sensitive cells. Our results indicate that SMKT kills sensitive S. cerevisiae by interacting with the yeast cell membrane.

  13. Interactions of the antiviral and antiparkinson agent amantadine with lipid membranes and human erythrocytes.

    PubMed

    Suwalsky, Mario; Jemiola-Rzeminska, Malgorzata; Altamirano, Mariella; Villena, Fernando; Dukes, Nathan; Strzalka, Kazimierz

    2015-07-01

    Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of amantadine to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, fluorescence spectroscopy and differential scanning calorimetry (DSC). In an attempt to further elucidate its effects on cell membranes, the present work also examined amantadine influence on the morphology of intact human erythrocytes by means of scanning electron microscopy (SEM). Results indicated that amantadine induced morphological changes to human erythrocytes and interacted in a concentration-dependent manner with DMPC bilayers in contrast to DMPE that was hardly affected by the presence of the drug. PMID:25899993

  14. Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

    PubMed

    Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Zhendre, Vanessa; Henry, Sarah; Thévenot, Julie; Dufourc, Erick J; Alves, Isabel D; Peruch, Frédéric

    2014-01-01

    The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles.

  15. Membrane-membrane interactions in a lipid-containing bacteriophage system. Progress report, October 1, 1980-September 30, 1981

    SciTech Connect

    Snipes, W

    1981-05-01

    Virus-cell interactions and the mechanism of viral entry have been the major focal points of this research. A method of analysis was perfected to investigate the entry process for herpes simplex virus. This technique makes use of a photosensitizing dye, FITC, that covalently binds to viral envelope proteins. Treated virions remain photosensitive until the envelope is shed during the process of infection. Our data strongly support an entry mechanism in which the viral envelope fuses with the cell plasma membrane. Other related projects have involved studies of the virucidal properties of retinoids, plaque development characteristics for viruses surviving treatment with membrane perturbers, and a large plaque effect that occurs when virus are plated on cells pretreated with uv light. In addition, we have characterized a new bacteriophage, investigated the interactions of divalent cations and proteins with phospholipid vesicles, extended our studies of the effects of hydrophobic photosensitizers on cell membranes, and used the spin-trapping technique to elucidate the reaction mechanism for an enzyme-like activity in soil extracts.

  16. Coarse-Grained Molecular Dynamics Simulations of Membrane-Trehalose Interactions.

    PubMed

    Kapla, Jon; Stevensson, Baltzar; Maliniak, Arnold

    2016-09-15

    It is well established that trehalose (TRH) affects the physical properties of lipid bilayers and stabilizes biological membranes. We present molecular dynamics (MD) computer simulations to investigate the interactions between lipid membranes formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and TRH. Both atomistic and coarse-grained (CG) interaction models were employed, and the coarse graining of DMPC leads to a reduction in the acyl chain length corresponding to a 1,2-dilauroyl-sn-glycero-3-phosphocholine lipid (DLPC). Several modifications of the Martini interaction model, used for CG simulations, were implemented, resulting in different potentials of mean force (PMFs) for DMPC bilayer-TRH interactions. These PMFs were subsequently used in a simple two-site analytical model for the description of sugar binding at the membrane interface. In contrast to that in atomistic MD simulations, the binding in the CG model was not in agreement with the two-site model. Our interpretation is that the interaction balance, involving water, TRH, and lipids, in the CG systems needs further tuning of the force-field parameters. The area per lipid is only weakly affected by TRH concentration, whereas the compressibility modulus related to the fluctuations of the membrane increases with an increase in TRH content. In agreement with experimental findings, the bending modulus is not affected by the inclusion of TRH. The important aspects of lipid bilayer interactions with biomolecules are membrane curvature generation and sensing. In the present investigation, membrane curvature is generated by artificial buckling of the bilayer in one dimension. It turns out that TRH prefers the regions with the highest curvature, which enables the most favorable situation for lipid-sugar interactions. PMID:27530142

  17. Wrapping of nanoparticles by the cell membrane: the role of interactions between the nanoparticles.

    PubMed

    Tang, Huayuan; Ye, Hongfei; Zhang, Hongwu; Zheng, Yonggang

    2015-11-28

    A fundamental understanding of the interactions between nanoparticles (NPs) and the cell membrane is essential to improve the performance of the NP-based biomedical applications and assess the potential toxicity of NPs. Despite the great progress in understanding the interaction between individual NP and the membrane, little is known about the interaction between multiple NPs and the membrane. In this work, we investigate the wrapping of two parallel elongated NPs by the membrane, taking the NP-NP electrostatic interaction and van der Waals (vdW) interaction into consideration. Three types of NPs, namely the rigid NPs with circular and elliptic cross-sections and the deformable NPs, are systematically investigated. The results show that the electrostatic interaction would enhance the tendency of the independent wrapping and inhibit the rotation of the elongated and equally charged NPs with elliptic cross-sections. Under the vdW interaction, the competition of the NP-NP adhesion and the membrane elastic energies with the NP-membrane adhesion energy leads the NPs to be wrapped cooperatively or independently. For the system with elongated NPs with elliptic cross-sections, the NPs are more likely to be wrapped independently as the shapes become more anisotropic and the NPs would rotate to contact each other with the flat sides in the cooperative wrapping configuration. Moreover, the soft NPs are more likely to be wrapped cooperatively compared with the stiff NPs. These results may provide guidelines to control the internalization pathway of NPs and improve the efficiency of NP-based drug delivery systems.

  18. Wrapping of nanoparticles by the cell membrane: the role of interactions between the nanoparticles.

    PubMed

    Tang, Huayuan; Ye, Hongfei; Zhang, Hongwu; Zheng, Yonggang

    2015-11-28

    A fundamental understanding of the interactions between nanoparticles (NPs) and the cell membrane is essential to improve the performance of the NP-based biomedical applications and assess the potential toxicity of NPs. Despite the great progress in understanding the interaction between individual NP and the membrane, little is known about the interaction between multiple NPs and the membrane. In this work, we investigate the wrapping of two parallel elongated NPs by the membrane, taking the NP-NP electrostatic interaction and van der Waals (vdW) interaction into consideration. Three types of NPs, namely the rigid NPs with circular and elliptic cross-sections and the deformable NPs, are systematically investigated. The results show that the electrostatic interaction would enhance the tendency of the independent wrapping and inhibit the rotation of the elongated and equally charged NPs with elliptic cross-sections. Under the vdW interaction, the competition of the NP-NP adhesion and the membrane elastic energies with the NP-membrane adhesion energy leads the NPs to be wrapped cooperatively or independently. For the system with elongated NPs with elliptic cross-sections, the NPs are more likely to be wrapped independently as the shapes become more anisotropic and the NPs would rotate to contact each other with the flat sides in the cooperative wrapping configuration. Moreover, the soft NPs are more likely to be wrapped cooperatively compared with the stiff NPs. These results may provide guidelines to control the internalization pathway of NPs and improve the efficiency of NP-based drug delivery systems. PMID:26381589

  19. Protein–protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins

    PubMed Central

    Kleanthous, Colin; Rassam, Patrice; Baumann, Christoph G

    2015-01-01

    It has until recently been unclear whether outer membrane proteins (OMPs) of Gram-negative bacteria are organized or distributed randomly. Studies now suggest promiscuous protein–protein interactions (PPIs) between β-barrel OMPs in Escherichia coli govern their local and global dynamics, engender spatiotemporal patterning of the outer membrane into micro-domains and are the basis of β-barrel protein turnover. We contextualize these latest advances, speculate on areas of bacterial cell biology that might be influenced by the organization of OMPs into supramolecular assemblies, and highlight the new questions and controversies this revised view of the bacterial outer membrane raises. PMID:26629934

  20. Tamoxifen-model membrane interactions: an FT-IR study

    NASA Astrophysics Data System (ADS)

    Boyar, Handan; Severcan, Feride

    1997-06-01

    The temperature- and concentration-induced effects of tamoxifen (TAM) on dipalmitoyl phosphatidylcholine (DPPC) model membranes were investigated by the Fourier transform-infrared (FT-IR) spectroscopic technique. An investigation of the C-H stretching region and the CO mode reveals that the inclusion of TAM changes the physical properties of the DPPC multibilayers by (i) shifting the main phase transition to lower temperatures; (ii) broadening the transition profile slightly; (iii) disordering the system in the gel and in the liquid crystalline phases; (iv) increasing the dynamics in the gel phase and decreasing the dynamics of the acyl chains in the liquid crystalline phase; (v) increasing the mobility of the terminal methyl group region of the bilayer in the gel phase and decreasing it in the liquid crystalline phase; (vi) increasing the frequency of the CO stretching mode both in the gel and in the liquid crystalline phases, i.e. non-bonding with carbonyl groups.

  1. Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes

    PubMed Central

    Dalton, Amanda K.; Murray, Paul S.; Murray, Diana; Vogt, Volker M.

    2005-01-01

    The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo. PMID:15858007

  2. Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast

    PubMed Central

    Klecker, Till; Wemmer, Megan; Haag, Mathias; Weig, Alfons; Böckler, Stefan; Langer, Thomas; Nunnari, Jodi; Westermann, Benedikt

    2015-01-01

    Membrane homeostasis affects mitochondrial dynamics, morphology, and function. Here we report genetic and proteomic data that reveal multiple interactions of Mdm33, a protein essential for normal mitochondrial structure, with components of phospholipid metabolism and mitochondrial inner membrane homeostasis. We screened for suppressors of MDM33 overexpression-induced growth arrest and isolated binding partners by immunoprecipitation of cross-linked cell extracts. These approaches revealed genetic and proteomic interactions of Mdm33 with prohibitins, Phb1 and Phb2, which are key components of mitochondrial inner membrane homeostasis. Lipid profiling by mass spectrometry of mitochondria isolated from Mdm33-overexpressing cells revealed that high levels of Mdm33 affect the levels of phosphatidylethanolamine and cardiolipin, the two key inner membrane phospholipids. Furthermore, we show that cells lacking Mdm33 show strongly decreased mitochondrial fission activity indicating that Mdm33 is critical for mitochondrial membrane dynamics. Our data suggest that MDM33 functionally interacts with components important for inner membrane homeostasis and thereby supports mitochondrial division. PMID:26669658

  3. Molecular determinants of α–synuclein mutants’ oligomerization and membrane interactions

    PubMed Central

    Tsigelny, Igor F.; Sharikov, Yuriy; Kouznetsova, Valentina L.; Greenberg, Jerry P.; Wrasidlo, Wolf; Overk, Cassia; Gonzalez, Tania; Trejo, Margarita; Spencer, Brian; Kosberg, Kori; Masliah, Eliezer

    2016-01-01

    Parkinson’s disease (PD) is associated with the formation of toxic α-synuclein oligomers and their penetration the cell membrane. Familial forms of PD are caused by the point mutations A53T, A30P, E46K, and H50Q. Artificial point mutations E35K and E57K also increase oligomerization and pore formation. We generated structural conformations of α-synuclein and the abovementioned mutants using molecular dynamics. We elucidated four main regions in these conformers contacting the membrane and found that the region including residues 37–45 (Zone2) may have maximum membrane penetration. E57K mutant had the highest rate of interaction with the membrane by Zone2, followed by A53T, E46K, E35K mutants, and wt α-synuclein. The mutant A30P had the smallest percentage of conformers that contact the membrane than all other mutants and wt α-synuclein. These results were confirmed by experiments. We identified the key amino acids that can interact with the membrane (Y38, E62, and N65 (1st hydrophilic layer); E104, E105, and D115 (2nd hydrophilic layer), and V15 and V26 (central hydrophobic layer)) and the residues that are involved in the interprotein contacts (L38, V48, V49, Q62, and T64). Understanding the molecular interactions of α-synuclein mutants is important for the design of compounds blocking the formation of toxic oligomers. PMID:25561023

  4. Screening membrane interactions of pesticides by cells decorated with chromatic polymer nanopatches.

    PubMed

    Mech, Agnieszka; Orynbayeva, Zulfiya; Irgebayev, Kaiyr; Kolusheva, Sofiya; Jelinek, Raz

    2009-01-01

    Elucidating the factors contributing to the cell toxicity of pesticides and other environmentally sensitive small molecules is critical for evaluation of their health impacts and for understanding the biological processes that they affect. Disruption and permeation of the plasma membrane, which constitutes the critical interface between the cell and its environment, are recognized initiators of cytotoxicity. We present a new approach for predicting pesticide cytotoxicity through rapid screening of membrane interactions of pesticides using a recently developed live-cell chromatic sensor. The sensing platform comprises living mammalian cells labeled with polydiacetylene (PDA), a chromatic polymer that undergoes intense fluorescence transformations induced by structural perturbations of the membrane bilayer. Within a short time after the addition of membrane-interacting tested compounds to the labeled cells, the PDA patches emit high fluorescence, which can be monitored by conventional spectroscopy and microscopy apparatuses. The chromatic technology facilitates rapid evaluation of membrane activity of pesticide compounds and is capable of distinguishing between toxic effects associated with membrane interactions vs intracellular mechanisms.

  5. Minimalist Model Systems Reveal Similarities and Differences between Membrane Interaction Modes of MCL1 and BAK*

    PubMed Central

    Landeta, Olatz; Landajuela, Ane; Garcia-Saez, Ana; Basañez, Gorka

    2015-01-01

    Proteins belonging to the BCL2 family are key modulators of apoptosis that establish a complex network of interactions among themselves and with other cellular factors to regulate cell fate. It is well established that mitochondrial membranes are the main locus of action of all BCL2 family proteins, but it is difficult to obtain a precise view of how BCL2 family members operate at the native mitochondrial membrane environment during apoptosis. Here, we used minimalist model systems and multiple fluorescence-based techniques to examine selected membrane activities of MCL1 and BAK under apoptotic-like conditions. We show that three distinct apoptosis-related factors (i.e. the BCL2 homology 3 ligand cBID, the mitochondrion-specific lipid cardiolipin, and membrane geometrical curvature) all promote membrane association of BCL2-like structural folds belonging to both MCL1 and BAK. However, at the same time, the two proteins exhibited distinguishing features in their membrane association modes under apoptotic-like conditions. In addition, scanning fluorescence cross-correlation spectroscopy and FRET measurements revealed that the BCL2-like structural fold of MCL1, but not that of BAK, forms stable heterodimeric complexes with cBID in a manner adjustable by membrane cardiolipin content and curvature degree. Our results add significantly to a growing body of evidence indicating that the mitochondrial membrane environment plays a complex and active role in the mode of action of BCL2 family proteins. PMID:25987560

  6. PTEN interaction with tethered bilayer lipid membranes containing PI(4,5)P2

    NASA Astrophysics Data System (ADS)

    Moldovan, R.; Shenoy, S.; Shekhar, P.; Kalinowski, A.; Gericke, A.; Heinrich, F.; Loesche, M.

    2009-03-01

    Synthetic lipid membrane models are frequently used for the study of biophysical processes at cell membranes. We use a robust membrane model, the tethered bilayer lipid membrane (tBLM), based on a (C14)2-(PEO)6-thiol anchor, WC14 [1]. Such membranes can be prepared to contain single phospholipids or complex lipid mixtures [2], including functional lipids involved in cell signaling, such as the highly charged phosphatidylinositol phosphates (PIPs). To study the interaction between the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) and model membranes we have incorporated phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in tBLMs and use fluorescence correlation spectroscopy (FCS), neutron reflectometry (NR) and surface plasmon resonance (SPR) for their characterization. NR shows that tBLMs formed with PI(4,5)P2 are complete. FCS of labeled PI(4,5)P2 shows that diffusion occurs at the time scale characteristic of membrane-incorporated lipid. Finally, SPR shows specific binding of PTEN to the model membrane thus confirming the incorporation of PI(4,5)P2 into the tBLM. [1] McGillivray et al, Biointerphases 2, 21-33 (2007) [2] Heinrich et al, Langmuir, submitted

  7. High-performance reverse osmosis CNT/polyamide nanocomposite membrane by controlled interfacial interactions.

    PubMed

    Kim, Hee Joong; Choi, Kwonyong; Baek, Youngbin; Kim, Dong-Gyun; Shim, Jimin; Yoon, Jeyong; Lee, Jong-Chan

    2014-02-26

    Polyamide reverse osmosis (RO) membranes with carbon nanotubes (CNTs) are prepared by interfacial polymerization using trimesoyl chloride (TMC) solutions in n-hexane and aqueous solutions of m-phenylenediamine (MPD) containing functionalized CNTs. The functionalized CNTs are prepared by the reactions of pristine CNTs with acid mixture (sulfuric acid and nitric acid of 3:1 volume ratio) by varying amounts of acid, reaction temperature, and reaction time. CNTs prepared by an optimized reaction condition are found to be well-dispersed in the polyamide layer, which is confirmed from atomic force microscopy, scanning electron microscopy, and Raman spectroscopy studies. The polyamide RO membranes containing well-dispersed CNTs exhibit larger water flux values than polyamide membrane prepared without any CNTs, although the salt rejection values of these membranes are close. Furthermore, the durability and chemical resistance against NaCl solutions of the membranes containing CNTs are found to be improved compared with those of the membrane without CNTs. The high membrane performance (high water flux and salt rejection) and the improved stability of the polyamide membranes containing CNTs are ascribed to the hydrophobic nanochannels of CNTs and well-dispersed states in the polyamide layers formed through the interactions between CNTs and polyamide in the active layers.

  8. The self-interaction of a nodavirus replicase is enhanced by mitochondrial membrane lipids.

    PubMed

    Qiu, Yang; Wang, Zhaowei; Liu, Yongxiang; Han, Yajuan; Miao, Meng; Qi, Nan; Yang, Jie; Xia, Hongjie; Li, Xiaofeng; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2014-01-01

    RNA replication of positive-strand (+)RNA viruses requires the protein-protein interactions among viral replicases and the association of viral replicases with intracellular membranes. Protein A from Wuhan nodavirus (WhNV), which closely associate with mitochondrial membranes, is the sole replicase required for viral RNA replication. Here, we studied the direct effects of mitochondrial membrane lipids (MMLs) on WhNV protein A activity in vitro. Our investigations revealed the self-interaction of WhNV protein A is accomplished via two different patterns (i.e., homotypic and heterotypic self-interactions via different interfaces). MMLs stimulated the protein A self-interaction, and this stimulation exhibited selectivity for specific phospholipids. Moreover, we found that specific phospholipids differently favor the two self-interaction patterns. Furthermore, manipulating specific phospholipid metabolism affected protein A self-interaction and the activity of protein A to replicate RNA in cells. Taken together, our findings reveal the direct effects of membrane lipids on a nodaviral RNA replicase. PMID:24586921

  9. The Self-Interaction of a Nodavirus Replicase Is Enhanced by Mitochondrial Membrane Lipids

    PubMed Central

    Qiu, Yang; Wang, Zhaowei; Liu, Yongxiang; Han, Yajuan; Miao, Meng; Qi, Nan; Yang, Jie; Xia, Hongjie; Li, Xiaofeng; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2014-01-01

    RNA replication of positive-strand (+)RNA viruses requires the protein-protein interactions among viral replicases and the association of viral replicases with intracellular membranes. Protein A from Wuhan nodavirus (WhNV), which closely associate with mitochondrial membranes, is the sole replicase required for viral RNA replication. Here, we studied the direct effects of mitochondrial membrane lipids (MMLs) on WhNV protein A activity in vitro. Our investigations revealed the self-interaction of WhNV protein A is accomplished via two different patterns (i.e., homotypic and heterotypic self-interactions via different interfaces). MMLs stimulated the protein A self-interaction, and this stimulation exhibited selectivity for specific phospholipids. Moreover, we found that specific phospholipids differently favor the two self-interaction patterns. Furthermore, manipulating specific phospholipid metabolism affected protein A self-interaction and the activity of protein A to replicate RNA in cells. Taken together, our findings reveal the direct effects of membrane lipids on a nodaviral RNA replicase. PMID:24586921

  10. Effect of cholesterol on the interaction of the HIV GP41 fusion peptide with model membranes. Importance of the membrane dipole potential.

    PubMed

    Buzón, Víctor; Cladera, Josep

    2006-12-26

    Fusion of viral and cell membranes is a key event in the process by which the human immunodeficiency virus (HIV) enters the target cell. Membrane fusion is facilitated by the interaction of the viral gp41 fusion peptide with the cell membrane. Using synthetic peptides and model membrane systems, it has been established that the sequence of events implies the binding of the peptide to the membrane, followed by a conformational change (transformation of unordered and helical structures into beta-aggregates) which precedes lipid mixing. It is known that this process can be influenced by the membrane lipid composition. In the present work we have undertaken a systematic study in order to determine the influence of cholesterol (abundant in the viral membrane) in the sequence of events leading to lipid mixing. Besides its effect on membrane fluidity, cholesterol can affect a less known physical parameter, the membrane dipole potential. Using the dipole potential fluorescent sensor di-8-ANEPPS together with other biophysical techniques, we show that cholesterol increases the affinity of the fusion peptide for the model membranes, and although it lowers the extent of lipid mixing, it increases the mixing rate. The influence of cholesterol on the peptide affinity and the lipid mixing rate are shown to be mainly due to its influence of the membrane dipole potential, whereas the lipid mixing extent and peptide conformational changes seem to be more dependent on other membrane parameters such as membrane fluidity and hydration.

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

    DOE PAGES

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

    2016-08-13

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

  12. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

    PubMed

    Lajko, Michelle; Haddad, Alexander F; Robinson, Carolyn A; Connolly, Sarah A

    2015-09-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

  13. Interaction study between maltose-modified PPI dendrimers and lipidic model membranes.

    PubMed

    Wrobel, Dominika; Appelhans, Dietmar; Signorelli, Marco; Wiesner, Brigitte; Fessas, Dimitrios; Scheler, Ulrich; Voit, Brigitte; Maly, Jan

    2015-07-01

    The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between

  14. Interaction study between maltose-modified PPI dendrimers and lipidic model membranes.

    PubMed

    Wrobel, Dominika; Appelhans, Dietmar; Signorelli, Marco; Wiesner, Brigitte; Fessas, Dimitrios; Scheler, Ulrich; Voit, Brigitte; Maly, Jan

    2015-07-01

    The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between

  15. Poisson-Boltzmann theory for membranes with mobile charged lipids and the pH-dependent interaction of a DNA molecule with a membrane.

    PubMed Central

    Fleck, Christian; Netz, Roland R; von Grünberg, Hans Hennig

    2002-01-01

    We consider a planar stiff model membrane consisting of mobile surface groups whose state of charge depends on the pH and the ionic composition of the adjacent electrolyte solution. To calculate the mean-field interaction potential between a charged object and such a model membrane, one needs to solve a Poisson-Boltzmann boundary value problem. We here derive and discuss the boundary condition at the membrane surface, a condition that is generally appropriate for biological membranes where two charge-regulating mechanisms are present at the same time: the pH-dependent chemical charge regulation and a regulation through the in-plane mobility of the surface groups. As an application of this general formalism, we consider the specific example of a single DNA molecule, approximated by a cylinder with smeared-out surface charges, interacting with such a model membrane. We study the effect that the two competing charge-regulating mechanisms have on the DNA/membrane interaction and the distribution of surface ions in the plane of the membrane. We find that, at short DNA-membrane distances, membrane fluidity can have a considerable impact on the DNA adsorption behavior and can lead to such counterintuitive phenomena as the adsorption of a negatively charged DNA onto a (on average) negatively charged membrane. PMID:11751297

  16. Interaction of a P. aeruginosa Quorum Sensing Signal with Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Morrison, Rebecca; Hall, Amelia; Hutchison, Ellen; Nguyen, Thuc; Cooley, Benjamin; Gordon, Vernita

    2011-03-01

    Bacteria use a signaling and regulatory system called ``quorum sensing'' to alter their gene expressions in response to the concentration of neighboring bacteria and to environmental conditions that make collective activity favorable for bacteria. P. aeruginosa is an opportunistic human pathogen that uses quorum sensing to govern processes such as virulence and biofilm formation. This organism's two main quorum sensing circuits use two different signaling molecules that are amphiphilic and differ primarily in the length of their hydrocarbon side chain and thus in their hydrophobic physical chemistry. How these physical chemistries govern the propagation and spatial localization of signals and thus of quorum sensing is not known. We present preliminary results showing that signals preferentially sequester to amphiphilic lipid membranes, which can act as reservoirs for signal. This is promising for future characterization of how the quorum sensing signals of many bacteria and yeast partition to spatially-differentiated amphiphilic environments, in a host or biofilm.

  17. Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

    PubMed

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2015-08-18

    Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

  18. A Laboratory Exercise to Illustrate Protein-Membrane Interactions

    ERIC Educational Resources Information Center

    Weers, Paul M. M.; Prenner, Elmar J.; Curic, Spomenka; Lohmeier-Vogel, Elke M.

    2016-01-01

    The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6-tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV,…

  19. Interaction of peptidomimetics with bilayer membranes: biophysical characterization and cellular uptake.

    PubMed

    Jing, Xiaona; Kasimova, Marina R; Simonsen, Anders H; Jorgensen, Lene; Malmsten, Martin; Franzyk, Henrik; Foged, Camilla; Nielsen, Hanne M

    2012-03-20

    Enzymatically stable cell-penetrating α-peptide/β-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (ΔG) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized ΔG being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, ΔG extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.

  20. Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.

    PubMed

    Gubbens, Jacob; Vader, Pieter; Damen, J Mirjam A; O'Flaherty, Martina C; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2007-05-01

    To analyze proteins interacting at the membrane interface, a phospholipid analogue was used with a photoactivatable headgroup (ASA-DLPE, N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) for selective cross-linking. The peripheral membrane protein cytochrome c from the inner mitochondrial membrane was rendered carbonate wash-resistant by cross-linking to ASA-DLPE in a model membrane system, validating our approach. Cross-link products of cytochrome c and its precursor apocytochrome c were demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), taking advantage of the intrinsic UV absorbance of the cross-linker. Application of the method to inner mitochondrial membranes from Saccharomyces cerevisae revealed cross-link products of both exogenously added apocytochrome c and endogenous proteins with molecular weights around 34 and 72 kDa. Liquid chromatograpy (LC)-MS/MS was performed to identify these proteins, resulting in a list of candidate proteins potentially cross-linked at the membrane interface. The approach described here provides methodology for capturing phospholipid-protein interactions in their native environment of the biomembrane using modern proteomics techniques.

  1. Optical Detection of Aqueous Phase Analytes via Host-Guest Interactions on a Lipid Membrane Surface

    SciTech Connect

    Sasaki, D.Y.; Waggoner, T.A.

    1999-01-11

    The organization and assembly of molecules in cellular membranes is orchestrated through the recognition and binding of specific chemical signals. A simplified version of the cellular membrane system has been developed using a synthetically prepared membrane receptor incorporated into a biologically derived lipid bilayer. Through an interplay of electrostatic and van der Wards interactions, aggregation or dispersion of molecular components could be executed on command using a specific chemical signal. A pyrene fluorophore was used as an optical probe to monitor the aggregational state of the membrane receptors in the bilayer matrix. The pyrene excimer emission to monomer emission (E/M) intensity ratio gave a relative assessment of the local concentration of receptors in the membrane. Bilayers were prepared with receptors selective for the divalent metal ions of copper, mercury, and lead. Addition of the metal ions produced a rapid dispersion of aggregated receptor components at nano- to micro-molar concentrations. The process was reversible by sequestering the metal ions with EDTA. Receptors for proteins and polyhistidine were also prepared and incorporated into phosphatidylcholine lipid bilayers. In this case, the guest molecules bound to the membrane through multiple points of interaction causing aggregation of initially dispersed receptor molecules. The rapid, selective, and sensitive fluorescence optical response of these lipid assemblies make them attractive in sensor applications for aqueous phase metal ions and polypeptides.

  2. [The study on the characters of membrane protein interaction and its network based on integrated intelligence method].

    PubMed

    Shen, Yizhen; Ding, Yongsheng; Hao, Kuangrong

    2011-08-01

    Membrane protein and its interaction network have become a novel research direction in bioinformatics. In this paper, a novel membrane protein interaction network simulator is proposed for system biology studies by integrated intelligence method including spectrum analysis, fuzzy K-Nearest Neighbor(KNN) algorithm and so on. We consider biological system as a set of active computational components interacting with each other and with the external environment. Then we can use the network simulator to construct membrane protein interaction networks. Based on the proposed approach, we found that the membrane protein interaction network almost has some dynamic and collective characteristics, such as small-world network, scale free distributing, and hierarchical module structure. These properties are similar to those of other extensively studied protein interaction networks. The present studies on the characteristics of the membrane protein interaction network will be valuable for its relatively biological and medical studies. PMID:21936357

  3. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  4. Influence of trifluoroethanol on membrane interfacial anchoring interactions of transmembrane alpha-helical peptides.

    PubMed

    Ozdirekcan, Suat; Nyholm, Thomas K M; Raja, Mobeen; Rijkers, Dirk T S; Liskamp, Rob M J; Killian, J Antoinette

    2008-02-15

    Interfacial anchoring interactions between aromatic amino acid residues and the lipid-water interface are believed to be important determinants for membrane protein structure and function. Thus, it is possible that molecules that partition into the lipid-water interface can influence membrane protein activity simply by interfering with these anchoring interactions. Here we tested this hypothesis by investigating the effects of 2,2,2-trifluoroethanol (TFE) on the interaction of a Trp-flanked synthetic transmembrane peptide (acetyl-GW(2)(LA)(8)LW(2)A-NH(2)) with model membranes of dimyristoylphosphatidylcholine. Two striking observations were made. First, using (2)H nuclear magnetic resonance on acyl chain deuterated lipids, we found that addition of 4 or 8 vol % of TFE completely abolishes the ability of the peptide to order and stretch the lipid acyl chains in these relatively thin bilayers. Second, we observed that addition of 8 vol % TFE reduces the tilt angle of the peptide from 5.3 degrees to 2.5 degrees, as measured by (2)H NMR on Ala-d(4) labeled peptides. The "straightening" of the peptide was accompanied by an increased exposure of Trp to the aqueous phase, as shown by Trp-fluorescence quenching experiments using acrylamide. The observation of a reduced tilt angle was surprising because we also found that TFE partioning results in a significant thinning of the membrane, which would increase the extent of hydrophobic mismatch. In contrast to the Trp-flanked peptide, no effect of TFE was observed on the interaction of a Lys-flanked analog (acetyl-GK(2)(LA)(8)LK(2)A-NH(2)) with the lipid bilayer. These results emphasize the importance of interfacial anchoring interactions for membrane organization and provide new insights into how molecules such as TFE that can act as anesthetics may affect the behavior of membrane proteins that are enriched in aromatic amino acids at the lipid-water interface.

  5. Preferential Solvation of a Highly Medium Responsive Pentacyanoferrate(II) Complex in Binary Solvent Mixtures: Understanding the Role of Dielectric Enrichment and the Specificity of Solute-Solvent Interactions.

    PubMed

    Papadakis, Raffaello

    2016-09-01

    In this work, the preferential solvation of an intensely solvatochromic ferrocyanide(II) dye involving a 4,4'-bipyridine-based ligand was examined in various binary solvent mixtures. Its solvatochromic behavior was rationalized in terms of specific and nonspecific solute-solvent interactions. An exceptional case of solvatochromic inversion was observed when going from alcohol/water to amide/water mixtures. These effects were quantified using Onsager's solvent polarity function. Furthermore, the sensitivity of the solvatochromism of the dye was determined using various solvatochromic parameters such as π* expressing the dipolarity/polarizability of solvents and α expressing the hydrogen-bond-donor acidity of solvents. This analysis was useful for the rationalization of the selective solvation phenomena occurring in the three types of alcohol/water and amide/water mixtures studied. Furthermore, two preferential solvation models were employed for the interpretation of the experimental spectral results in binary solvent mixtures, namely, the model of Suppan on dielectric enrichment [J. Chem. Soc. Faraday Trans. 1 1987, 83, 495-509] and the model of Bosch, Rosés, and co-workers [J. Chem. Soc., Perkin Trans. 2, 1995, 8, 1607-1615]. The first model successfully predicted the charge transfer energies of the dye in formamide/water and N-methylformamide/water mixtures, but in the case of MeOH/water mixtures, the prediction was less accurate because of the significant contribution of specific solute-solvent interactions in that case. The second model gave more insights for both specific solute-solvent as well as solvent-solvent interactions in the cybotactic region. The role of dielectric enrichment and specific interactions was discussed based on the findings. PMID:27525362

  6. Preferential Solvation of a Highly Medium Responsive Pentacyanoferrate(II) Complex in Binary Solvent Mixtures: Understanding the Role of Dielectric Enrichment and the Specificity of Solute-Solvent Interactions.

    PubMed

    Papadakis, Raffaello

    2016-09-01

    In this work, the preferential solvation of an intensely solvatochromic ferrocyanide(II) dye involving a 4,4'-bipyridine-based ligand was examined in various binary solvent mixtures. Its solvatochromic behavior was rationalized in terms of specific and nonspecific solute-solvent interactions. An exceptional case of solvatochromic inversion was observed when going from alcohol/water to amide/water mixtures. These effects were quantified using Onsager's solvent polarity function. Furthermore, the sensitivity of the solvatochromism of the dye was determined using various solvatochromic parameters such as π* expressing the dipolarity/polarizability of solvents and α expressing the hydrogen-bond-donor acidity of solvents. This analysis was useful for the rationalization of the selective solvation phenomena occurring in the three types of alcohol/water and amide/water mixtures studied. Furthermore, two preferential solvation models were employed for the interpretation of the experimental spectral results in binary solvent mixtures, namely, the model of Suppan on dielectric enrichment [J. Chem. Soc. Faraday Trans. 1 1987, 83, 495-509] and the model of Bosch, Rosés, and co-workers [J. Chem. Soc., Perkin Trans. 2, 1995, 8, 1607-1615]. The first model successfully predicted the charge transfer energies of the dye in formamide/water and N-methylformamide/water mixtures, but in the case of MeOH/water mixtures, the prediction was less accurate because of the significant contribution of specific solute-solvent interactions in that case. The second model gave more insights for both specific solute-solvent as well as solvent-solvent interactions in the cybotactic region. The role of dielectric enrichment and specific interactions was discussed based on the findings.

  7. Fluctuation Limit for Interacting Diffusions with Partial Annihilations Through Membranes

    NASA Astrophysics Data System (ADS)

    Chen, Zhen-Qing; Fan, Wai-Tong Louis

    2016-08-01

    We study fluctuations of the empirical processes of a non-equilibrium interacting particle system consisting of two species over a domain that is recently introduced in Chen and Fan (Ann Probab, to appear) and establish its functional central limit theorem. This fluctuation limit is a distribution-valued Gaussian Markov process which can be represented as a mild solution of a stochastic partial differential equation. The drift of our fluctuation limit involves a new partial differential equation with nonlinear coupled term on the interface that characterized the hydrodynamic limit of the system. The covariance structure of the Gaussian part consists two parts, one involving the spatial motion of the particles inside the domain and other involving a boundary integral term that captures the boundary interactions between two species. The key is to show that the Boltzmann-Gibbs principle holds for our non-equilibrium system. Our proof relies on generalizing the usual correlation functions to the join correlations at two different times.

  8. The anticancer drug chlorambucil interacts with the human erythrocyte membrane and model phospholipid bilayers.

    PubMed

    Suwalsky, M; Hernández, P; Villena, F; Sotomayor, C P

    1999-12-01

    The plasma membrane has gained increasing attention as a possible target of antitumor drugs. It has been reported that they act as growth factor antagonists, growth factor receptor blockers, interfere with mitogenic signal transduction or exert direct cytotoxic effects. Chlorambucil (4-[p-(bis[2-chloroethyl]amino)phenyl]butyric acid) is an alkylating agent widely used in the treatment of chronic lymphocytic leukaemia. Contradictory reports have been published concerning its interaction with cell membranes. Whereas a decrease in the fluidity of Ehrlich ascite tumor cells has been adduced, no evidences were found that chlorambucil changes membrane lipid fluidity and alkylating agents had effects in these systems even at highly toxic concentrations. Our results showed that chlorambucil at a dose equivalent to its therapeutical concentration in the plasma (3.6 microM) caused the human erythrocyte membrane to develop cup-shaped forms (stomatocytes). Accordingly to the bilayer couple hypothesis, this means that the drug is inserted into the inner monolayer of the erythrocyte membrane, a conclusion supported by X-ray diffraction performed on multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. It is concluded that the cytotoxic effect of chlorambucil might be due to alteration of the structure and therefore of the physiological properties of cell membranes such as fluidity, permeability, receptor and channel functions. PMID:10685501

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

    PubMed

    Swain, Jitendriya; Kumar Mishra, Ashok

    2015-09-10

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

  10. Interaction between Polyamines and Bacterial Outer Membranes as Investigated with Ion-Selective Electrodes

    PubMed Central

    Katsu, Takashi; Nakagawa, Hideki; Yasuda, Keiko

    2002-01-01

    We analyzed the interaction between polyamines and the outer membrane of Escherichia coli cells using potentiometric measurements with Ca2+, tetraphenylphosphonium (TPP+), and K+ electrodes. The Ca2+ electrode was used to examine the ability of the polyamines to release Ca2+ from the outer membrane. The TPP+ electrode was used to examine the ability to permeabilize the outer membrane, since the uptake of TPP+ was enhanced when the permeability barrier of the outer membrane was disrupted. The K+ electrode was used to examine permeabilization in the cytoplasmic membrane by monitoring the efflux of K+ in cytosol. Although Ca2+ release was remarkably enhanced by increasing the number of amino groups in polyamines, no TPP+ uptake was observed with polyamines of a simple structure, such as ethylenediamine, spermidine, and spermine. TPP+ uptake was observed when appropriate lipophilic moieties were further attached to the polyamines with three or four amino groups, indicating that the existence of bulky moieties as well as the number of amino groups is important to induce outer membrane permeabilization. Thus, 1-naphthylacetylspermine and N,N′-bis[6-[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine (methoctramine) were especially effective in increasing the permeability of the outer membrane of E. coli cells, being comparable to polymyxin B nonapeptide, a well-known cationic peptide showing such action. PMID:11897592

  11. High-resolution 2D NMR spectroscopy of bicelles to measure the membrane interaction of ligands.

    PubMed

    Dvinskikh, Sergey V; Dürr, Ulrich H N; Yamamoto, Kazutoshi; Ramamoorthy, Ayyalusamy

    2007-01-31

    Magnetically aligned bicelles are increasingly being used as model membranes in solution- and solid-state NMR studies of the structure, dynamics, topology, and interaction of membrane-associated peptides and proteins. These studies commonly utilize the PISEMA pulse sequence to measure dipolar coupling and chemical shift, the two key parameters used in subsequent structural analysis. In the present study, we demonstrate that the PISEMA and other rotating-frame pulse sequences are not suitable for the measurement of long-range heteronuclear dipolar couplings, and that they provide inaccurate values when multiple protons are coupled to a 13C nucleus. Furthermore, we demonstrate that a laboratory-frame separated-local-field experiment is capable of overcoming these difficulties in magnetically aligned bicelles. An extension of this approach to accurately measure 13C-31P and 1H-31P couplings from phospholipids, which are useful to understand the interaction of molecules with the membrane, is also described. In these 2D experiments, natural abundance 13C was observed from bicelles containing DMPC and DHPC lipid molecules. As a first application, these solid-state NMR approaches were utilized to probe the membrane interaction of an antidepressant molecule, desipramine, and its location in the membrane.

  12. A mirror code for protein-cholesterol interactions in the two leaflets of biological membranes

    PubMed Central

    Fantini, Jacques; Di Scala, Coralie; Evans, Luke S.; Williamson, Philip T. F.; Barrantes, Francisco J.

    2016-01-01

    Cholesterol controls the activity of a wide range of membrane receptors through specific interactions and identifying cholesterol recognition motifs is therefore critical for understanding signaling receptor function. The membrane-spanning domains of the paradigm neurotransmitter receptor for acetylcholine (AChR) display a series of cholesterol consensus domains (referred to as “CARC”). Here we use a combination of molecular modeling, lipid monolayer/mutational approaches and NMR spectroscopy to study the binding of cholesterol to a synthetic CARC peptide. The CARC-cholesterol interaction is of high affinity, lipid-specific, concentration-dependent, and sensitive to single-point mutations. The CARC motif is generally located in the outer membrane leaflet and its reverse sequence CRAC in the inner one. Their simultaneous presence within the same transmembrane domain obeys a “mirror code” controlling protein-cholesterol interactions in the outer and inner membrane leaflets. Deciphering this code enabled us to elaborate guidelines for the detection of cholesterol-binding motifs in any membrane protein. Several representative examples of neurotransmitter receptors and ABC transporters with the dual CARC/CRAC motifs are presented. The biological significance and potential clinical applications of the mirror code are discussed. PMID:26915987

  13. Glucocorticoid interactions with ethanol effects on synaptic plasma membranes: influence on [125I]calmodulin binding.

    PubMed

    Sze, P Y

    1996-02-01

    Ca(++)-dependent binding of calmodulin (CaM) to brain synaptic plasma membranes is known to be inhibited by ethanol and stimulated by glucocorticoids. These opposite neurochemical actions between ethanol and the steroids in vitro are consistent with glucocorticoid antagonism of ethanol-induced sedation reported to occur in vivo. The present study was undertaken to characterize the interactions of corticosterone with ethanol effects on [125I]CaM binding in synaptic plasma membranes. From the shift of concentration-response curves when corticosterone and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition occurred in an additive relationship over the range of their effective concentrations. From Scatchard analyses, ethanol-induced decrease in membrane affinity for [125I]CaM was antagonized by steroid-induced increase in the membrane affinity, indicating that the convergent event in their interaction was the alteration of membrane affinity for CaM. Glucocorticoid antagonism of ethanol inhibition of [125I]CaM binding exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. The demonstration that glucocorticoids antagonized the inhibition of CaM binding by ethanol provides support for the hypothesis that these steroids are among the endogenous factors that modulate neuronal sensitivity to ethanol.

  14. A possible molecular mechanism for the interaction of defensin with the sensory neuron membrane.

    PubMed

    Plakhova, V B; Shchegolev, B F; Rogachevskii, I V; Nozdrachev, A D; Krylov, B V; Podzorova, S A; Kokryakov, V N

    2002-01-01

    A local membrane potential clamping method was used to study the effects of defensin NP-1 on the membranes of rat spinal ganglion neurons. NP-1 led to decreases in the effective charge for the activation gating system. This process depended on the NP-1 concentration. Use of the Hill equation showed that Kd was 2.10(-12) M and the Hill coefficient was 0.9. The structure of the defensin molecule was optimized using quantum chemical calculations based on a molecular mechanics method. The results obtained from these calculations suggested that a single hydroxyl group directed towards the outer part of thedefensin molecule and forming the carboxyl group of amino acid Glu14 could form a hydrogen bond with the active center of the membrane receptor. This explains the experimentally observed 1:1 stoichiometry of the ligand-receptor binding interaction between the defensin and the sensory neuron membrane. PMID:12243262

  15. Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain.

    PubMed Central

    Scheiffele, P; Roth, M G; Simons, K

    1997-01-01

    Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes. PMID:9312009

  16. Characterization of the interaction of two oscillating bubbles near a thin elastic membrane

    NASA Astrophysics Data System (ADS)

    Aghdam, A. Hajizadeh; Farhangmehr, V.; Ohl, S. W.; Khoo, B. C.; Shervani-Tabar, M. T.

    2012-12-01

    Oscillating bubbles appear in the bodily fluid during many medical treatments, for example in Extracorporeal Shockwave Lithotripsy. We report a systematic study on the complex interaction between two such bubbles and an elastic membrane, which could be a biological membrane in the human body. We have grouped our analysis into similarly sized bubbles, and differently sized bubbles. All bubbles are created at the same time. For the similarly sized bubbles, it can be broadly characterized as the splitting up of two bubbles in vertical direction perpendicular to (vertical split) and at an angle to (oblique split) the membrane surface, jetting towards each other and bubble coalescence. For the two differently sized bubbles, there is the jetting towards or away from the large bubble for the small bubble and the `catapult' effect observed. The two bubbles dynamics depend on the relative bubble sizes, the distance from the membrane, and the inter-bubble distance.

  17. Analysis of the interactions of sulfur-containing amino acids in membrane proteins.

    PubMed

    Gómez-Tamayo, José C; Cordomí, Arnau; Olivella, Mireia; Mayol, Eduardo; Fourmy, Daniel; Pardo, Leonardo

    2016-08-01

    The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic-aromatic, aromatic-aliphatic or/and aliphatic-aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small-molecule model systems at the ab-initio level, predicts that Met-Met interactions are stronger than Met-Cys ≈ Met-Phe ≈ Cys-Phe interactions, stronger than Phe-Phe ≈ Phe-Leu interactions, stronger than the Met-Leu interaction, and stronger than Leu-Leu ≈ Cys-Leu interactions. These results show that sulfur-containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity.

  18. The Interaction of Polyglutamine Peptides with Lipid Membranes Is Regulated by Flanking Sequences Associated with Huntingtin*

    PubMed Central

    Burke, Kathleen A.; Kauffman, Karlina J.; Umbaugh, C. Samuel; Frey, Shelli L.; Legleiter, Justin

    2013-01-01

    Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain. PMID:23572526

  19. Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy.

    PubMed

    Arsov, Zoran; Quaroni, Luca

    2007-11-01

    By using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and curve fitting we have examined temperature dependence and composition dependence of the shape of the carbonyl band in phosphatidylcholine/cholesterol model membranes. Membranes were hydrated either in excess water or in excess deuterated water. The studied binary mixtures exhibit different lipid phases at appropriate temperature and amount of cholesterol, among them also the so-called liquid-ordered phase. The results confirm that cholesterol has a significant indirect influence on the carbonyl band through conformational and hydration effects. This influence was interpreted in view of the known temperature composition phase diagrams for inspected binary mixtures. In addition, direct interaction was observed, which could point to the presence of hydrogen bond between cholesterol and carbonyl group. This direct interaction, though weak, might play at least a partial role in the stabilization of cholesterol-rich lipid domains in model and biological membranes. PMID:17662974

  20. Ab initio MO studies of interaction mechanisms of Protein Kinase C with cell membranes

    NASA Astrophysics Data System (ADS)

    Tsuda, Ken-ichiro; Kaneko, Hiroki; Shimada, Jiro; Takada, Toshikazu

    2001-12-01

    Protein Kinase C (PKC) is a family of regulatory enzymes. It is considered that binding with phorbol ester which are PKC activators, increases affinity of PKC for the membranes and consequently induces its conformation change. Electrostatic interactions between PKC and the membrane is assumed to be important, and performed ab initio MO calculations of one domain of PKC consisting of 50 amino acids and its complex with the ester is performed to investigate how the electrostatic potential of PKC changes through docking with the substrate. From the calculation, it is shown that the electrostatic potential of PKC near the binding site is dramatically affected through the binding, suggesting attractive interactions with the cell membrane.

  1. Arsenite interactions with phospholipid bilayers as molecular models for the human erythrocyte membrane.

    PubMed

    Suwalsky, Mario; Rivera, Cecilia; Villena, Fernando; Sotomayor, Carlos P; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

    2007-04-01

    There are scanty reports concerning the effects of arsenic compounds on the structure and functions of cell membranes. With the aim to better understand the molecular mechanisms of the interaction of arsenite with cell membranes we have utilized bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of arsenite to perturb the bilayer structures was determined by X-ray diffraction and fluorescence spectroscopy, whilst the modification of their thermotropic behaviour was followed by differential scanning calorimetry (DSC). The experiments carried out by X-ray diffraction and calorimetry clearly indicated that NaAsO(2) interacted with DMPE and modified its thermotropic behaviour. No such information has been so far reported in the literature. PMID:17175091

  2. Interaction of the antimicrobial peptide gomesin with model membranes: a calorimetric study.

    PubMed

    Domingues, Tatiana M; Mattei, Bruno; Seelig, Joachim; Perez, Katia R; Miranda, Antonio; Riske, Karin A

    2013-07-01

    Gomesin is a potent cationic antimicrobial peptide (z = +6) isolated from the Brazilian spider Acanthoscurria gomesiana . The interaction of gomesin with large unilamellar vesicles composed of a 1:1 mixture of zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) phospholipids is studied with isothermal titration calorimetry (ITC). In parallel, light scattering and optical microscopy are used to assess peptide-induced vesicle aggregation. The ability of gomesin to permeabilize the membrane is examined with fluorescence spectroscopy of the leakage of 5,6-carboxyfluorescein (CF). Vesicles coated with 3 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG) lipids are also investigated to assess the influence of peptide-induced vesicle aggregation in the activity of gomesin. The ITC and light scattering titrations are done in two ways: lipid into peptide and peptide into lipid injections. Although some differences arise between the two setups, the basic interaction of gomesin with anionic vesicles is preserved. A surface partition model combined with the Gouy-Chapman theory is put forward to fit the ITC results. The intrinsic binding constant of gomesin is found to be K ≈ 10(3) M(-1). The interaction of gomesin with anionic membranes is highly exothermic and enthalpy-driven. Binding of gomesin is virtually always accompanied by vesicle aggregation and changes in membrane permeability, leading to CF leakage. Addition of PE-PEG to the membrane strongly attenuates vesicle aggregation but does not significantly change the mode of action of gomesin. The results point to a strong interaction of gomesin with the membrane surface, causing membrane rupture without a deep penetration into the bilayer core.

  3. Contribution of membrane permeability and unstirred layer diffusion to nitric oxide-red blood cell interaction

    PubMed Central

    Deonikar, Prabhakar; Kavdia, Mahendra

    2012-01-01

    Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra- cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO-RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (Pm), hematocrit (Hct) and NO-Hb reaction rate constants on NO-RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO-RBC interactions. The extracellular diffusion contributed 70–90% of total transport resistance for Pm >1 cm/s whereas membrane resistance accounts for 50–75% of total transport resistance for Pm < 0.1 cm/s. We propose a narrow Pm range of 0.21–0.44 cm/s for 10–45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO-RBC interactions. PMID:23116664

  4. Interaction of the anticancer drug tamoxifen with the human erythrocyte membrane and molecular models.

    PubMed

    Suwalsky, M; Hernández, P; Villena, F; Aguilar, F; Sotomayor, C P

    1998-01-01

    Tamoxifen is a non steroidal antiestrogen drug extensively used in the prevention and treatment of hormone-dependent breast cancer. To evaluate its perturbing effect upon cell membranes it was made to interact with human erythrocytes and molecular models. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipids classes located in the outer and inner leaflets of the erythrocyte membrane, respectively. Experiments by fluorescence spectroscopy showed that tamoxifen interacted with DMPC vesicles fluidizing both its polar head and acyl chain regions. These results were confirmed by X-ray diffraction which indicated that tamoxifen perturbed the same regions of the lipid. However, it did not cause any significant structural perturbation to DMPE bilayers. The examination by electron microscopy of human erythrocytes incubated with tamoxifen revealed that they changed their normal discoid shape to stomatocytes. According to the bilayer couple hypothesis, this result means that the drug is inserted in the inner leaflet of the erythrocyte membrane. Given the fact that tamoxifen did not interact with DMPE, it is concluded that it interacted with a protein located in the cytoplasmic moiety of the erythrocyte membrane. PMID:9618934

  5. Molecular Dynamics Study on the Biophysical Interactions of Seven Green Tea Catechins with Cell Membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular dynamics simulations were performed to study the interactions of bioactive catechins (flavonoids) commonly found in green tea with lipid bilayers, as model for cell membranes. Previously, a number of experimental studies rationalized catechin’s anticarcinogenic, antibacterial, and other be...

  6. Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity.

    PubMed

    Rajan, Robin; Hayashi, Fumiaki; Nagashima, Toshio; Matsumura, Kazuaki

    2016-05-01

    Cryopreservation enables long-term preservation of cells at ultralow temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA) (poly(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly(sulfobetaine) (SPB) and poly(carboxymethyl betaine) (CMB), and compared their efficiency with poly(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Additionally, cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency. PMID:27077533

  7. Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane.

    PubMed

    Rodi, P M; Bocco Gianello, M D; Corregido, M C; Gennaro, A M

    2014-03-01

    The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a "facial" detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K=0.06mM(-1)), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.

  8. New insights into water-phospholipid model membrane interactions.

    PubMed

    Milhaud, Jeannine

    2004-05-27

    Modulating the relative humidity (RH) of the ambient gas phase of a phospholipid/water sample for modifying the activity of phospholipid-sorbed water [humidity-controlled osmotic stress methods, J. Chem. Phys. 92 (1990) 4519 and J. Phys. Chem. 96 (1992) 446] has opened a new field of research of paramount importance. New types of phase transitions, occurring at specific values of this activity, have been then disclosed. Hence, it is become recognized that this activity, like the temperature T, is an intensive parameter of the thermodynamical state of these samples. This state can be therefore changed (phase transition) either, by modulating T at a given water activity (a given hydration level), or, by modulating the water activity, at a given T. The underlying mechanisms of these two types of transition differ, especially when they appear as disorderings of fatty chains. In lyotropic transitions, this disordering follows from two thermodynamical laws. First, acting on the activity (the chemical potential) of water external to a phospholipid/water sample, a transbilayer gradient of water chemical potential is created, leading to a transbilayer flux of water (Fick's law). Second, water molecules present within the hydrocarbon region of this phospholipid bilayer interact with phospholipid molecules through their chemical potential (Gibbs-Duhem relation): the conformational state of fatty chains (the thermodynamical state of the phospholipid molecules) changes. This process is slow, as revealed by osmotic stress time-resolved experiments. In thermal chain-melting transitions, the first rapid step is the disordering of fatty chains of a fraction of phospholipid molecules. It occurs a few degrees before the main transition temperature, T(m), during the pretransition and the sub-main transition. The second step, less rapid, is the redistribution of water molecules between the different parts of the sample, as revealed by T-jump time-resolved experiments. Finally, in

  9. NMR study of chloride ion interactions with thylakoid membranes

    PubMed Central

    Baianu, I. C.; Critchley, C.; Govindjee; Gutowsky, H. S.

    1984-01-01

    The role of Cl- in photosynthetic O2 evolution has been investigated by observing the 35Cl NMR linewidth under a variety of conditions in aqueous suspensions of chloroplasts, primarily for the halophytes Avicennia germinans, Avicennia marina, and Aster tripolium but also for spinach. The line broadening shows there is weak, ionic binding of Cl- to thylakoids, the bound Cl- exchanging rapidly (>>104 sec-1) with free Cl- in solution. The binding is necessary for O2 evolution to occur. Michaelis-Menten constants obtained from the Cl- dependence of the O2 evolution rate are ≈15-70 mM for the halophytes compared with 0.6 mM for spinach (0.5 mM with Br-). There appear to be two types of Cl- binding sites in halophytes, of which the stronger is the activator, at lower [Cl-], of O2 evolution. The 35Cl line broadening includes a nonspecific interaction, which becomes apparent at high Cl- concentrations (≥0.5 M). PMID:16593474

  10. Interaction of lectins with membrane receptors on erythrocyte surfaces.

    PubMed

    Sung, L A; Kabat, E A; Chien, S

    1985-08-01

    The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.

  11. Perturbation of polysome - membrane interaction by aflatoxin B/sub 1/

    SciTech Connect

    Erki, L.; Patterson, W.J. Jr.; delRosario, R.; Devlin, T.M.; Ch'ih, J.J.

    1987-05-01

    In this study AFB/sup 1/ was used to assess the interaction between polysomes and the membrane. Hepatocytes treated with 50 ..mu..M AFB/sub 1/ showed no significant quantitative changes in phospholipid, protein, and RNA as compared with untreated cells. Mitochondrial and microsomal fractions isolated from AFB/sub 1/ treated cells showed 10-20% increases in protein and phospholipid. Determinations of membrane components of smooth endoplasmic reticulum (SER) and RER isolated from AFB/sub 1/ treated S-10 reaction gave similar results. To study AFB/sub 1/ activation and modification along with polysome and membrane interactions, SER or RER was incubated in the presence or absence of polysomes. In the presence of 0.25, 1 and 3 mg polysomes, (/sup 3/H)AFB/sub 1/ activation was inhibited by 28, 38, and 59%, respectively. When SER was incubated with (/sup 3/H)AFB/sub 1/ for 20 min in the absence of polysomes, the subsequent addition of varying amounts of polysomes resulted in a concentration dependent modification of polysomes. This concentration dependent modification and inhibition of AFB/sub 1/ activation by polysomes and the lack of changes in membrane components by AFB/sub 1/ indicated that the membrane sites for AFB/sub 1/ activation and polysome attachment are in close proximity.

  12. Interactions between non-steroidal anti-inflammatory drugs and lipid membranes

    NASA Astrophysics Data System (ADS)

    Boggara, Mohan; Krishnamoorti, Ramanan

    2008-03-01

    Chronic usage of Non-steroidal anti-inflammatory drugs(NSAIDs) leads to gastrointestinal toxicity and clinical evidences point the cause to direct interactions between NSAIDs and phospholipid membranes. Also, NSAIDs pre-associated with phospholipid vesicles are shown to be safer and therapeutically more effective than unmodified ones. Our initial experiments and simulations on the partitioning of Aspirin and Ibuprofen clearly indicate role played by the drug structure in drug-membrane interactions. Those results motivated systematic molecular dynamics simulations of membranes with NSAIDs of different size, structure and pKa values. Our results suggest high partition coefficients for these NSAIDs in the membrane compared to water and thinning effect on the bilayer. Our small angle neutron scattering and reflectivity studies on DMPC-Ibuprofen systems indicate that the drug affects both ˜5 nm thick bilayer and overall ˜100 nm diameter vesicle, indicating that NSAIDs affect vesicles on various length scales. We will discuss the structural perturbations to membranes due to NSAIDs at clinically relevant molar ratios and their implications on the use of vesicles as delivery vehicles for NSAIDs.

  13. Contact studies of weak adhesive interactions in water with membrane enhanced surface acoustic wave analysis

    NASA Astrophysics Data System (ADS)

    Brass, David Alan

    The measurement of weak adhesive energies has previously been difficult to obtain. To measure these energies, I designed a technique that uses the combined sensitivities of both a quartz crystal resonator and the inflation of an elastomeric polymer membrane. The surfaces of the quartz crystal and/or the membrane are modified with water swollen polymer brushes, which are used to eliminate nonspecific adhesion. These brushes are then end-modified with adhesive functional groups. An analysis is developed for the frequency response of a quartz crystal resonator as the membrane layer is placed in contact with the surface of these swollen brushes. The shear wave generated at the resonator surface couples into the membrane layer with an efficiency that is strongly dependent on the thickness of the swollen brush layer. The calculated shift decreases substantially for increases in the brush thickness of ten to twenty nanometers, giving a net frequency response that is extremely sensitive to the degree of swelling of the brush. An optimum capping layer thickness is determined by balancing the resonant frequency shift against dissipative effects that weaken the crystal resonance. Detailed calculations are presented for the specific case of poly(ethylene glycol) (PEG) brushes swollen by water and capped by a poly(styrene-ethylene/butene-styrene) (SEBS) elastomeric, water-permeable membrane. These calculations show that the method is sensitive to the properties of the brush layer. This surface acoustic wave technique was coupled with an inflation method that enabled quantification of the adhesion between the membrane and the brush coated surface. This adhesive interaction is obtained from the contact angle made between the quartz and membrane surfaces and the tension on the membrane. An analysis of the membrane profile based on the numerical solution of the axisymmetric Laplace equation is developed and used to investigate both adhesive and non-adhesive situations with both an

  14. Insights into the membrane interaction mechanism and antibacterial properties of chensinin-1b.

    PubMed

    Sun, Yue; Dong, Weibing; Sun, Li; Ma, Lijie; Shang, Dejing

    2015-01-01

    Antimicrobial peptides (AMPs) with non-specific membrane disrupting activities are thought to exert their antimicrobial activity as a result of their cationicity, hydrophobicity and α-helical or β-sheet structures. Chensinin-1, a native peptide from skin secretions of Rana chensinensis, fails to manifest its desired biological properties because its low hydrophobic nature and an adopted random coil structure in a membrane-mimetic environment. In this study, chensinin-1b was designed by rearranging the amino acid sequence of its hydrophilic/polar residues on one face and its hydrophobic/nonpolar residues on the opposite face according to its helical diagram, and by replacing three Gly residues with three Trp residues. Introduction of Trp residues significantly promoted the binding of the peptide to the bacterial outer membrane and exerted bactericidal activity through cytoplasmic membrane damage. Chensinin-1b demonstrates higher antimicrobial activity and greater cell selectivity than its parent peptide, chensinin-1. The electrostatic interactions between chensinin-1b and lipopolysaccharide (LPS) may have facilitated the uptake of the peptide into Gram-negative cells and be also helpful to disrupt the bacterial cytoplasmic membrane, as evidenced by depolarisation of the membrane potential and leakage of calceins from the liposomes of Escherichia coli and Staphylococcus aureus. Chensinin-1b was also found to penetrate mouse skin and was also effective in vivo, as measured by hydroxyproline levels in a wound infection mouse model, and could therefore act as an anti-infective agent for wound healing.

  15. Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions*

    PubMed Central

    Henriques, Sónia Troeira; Huang, Yen-Hua; Castanho, Miguel A. R. B.; Bagatolli, Luis A.; Sonza, Secondo; Tachedjian, Gilda; Daly, Norelle L.; Craik, David J.

    2012-01-01

    Cyclotides are bioactive cyclic peptides isolated from plants that are characterized by a topologically complex structure and exceptional resistance to enzymatic or thermal degradation. With their sequence diversity, ultra-stable core structural motif, and range of bioactivities, cyclotides are regarded as a combinatorial peptide template with potential applications in drug design. The mode of action of cyclotides remains elusive, but all reported biological activities are consistent with a mechanism involving membrane interactions. In this study, a diverse set of cyclotides from the two major subfamilies, Möbius and bracelet, and an all-d mirror image form, were examined to determine their mode of action. Their lipid selectivity and membrane affinity were determined, as were their toxicities against a range of targets (red blood cells, bacteria, and HIV particles). Although they had different membrane-binding affinities, all of the tested cyclotides targeted membranes through binding to phospholipids containing phosphatidylethanolamine headgroups. Furthermore, the biological potency of the tested cyclotides broadly correlated with their ability to target and disrupt cell membranes. The finding that a broad range of cyclotides target a specific lipid suggests their categorization as a new lipid-binding protein family. Knowledge of their membrane specificity has the potential to assist in the design of novel drugs based on the cyclotide framework, perhaps allowing the targeting of peptide drugs to specific cell types. PMID:22854971

  16. Fluid-Structure interaction analysis and performance evaluation of a membrane blade

    NASA Astrophysics Data System (ADS)

    Saeedi, M.; Wüchner, R.; Bletzinger, K.-U.

    2016-09-01

    Examining the potential of a membrane blade concept is the goal of the current work. In the sailwing concept the surface of the wing, or the blade in this case, is made from pre-tensioned membranes which meet at the pre-tensioned edge cable at the trailing edge. Because of the dependency between membrane deformation and applied aerodynamic load, two-way coupled fluid-structure interaction analysis is necessary for evaluation of the aerodynamic performance of such a configuration. The in-house finite element based structural solver, CARAT++, is coupled with OpenFOAM in order to tackle the multi-physics problem. The main aerodynamic characteristics of the membrane blade including lift coefficient, drag coefficient and lift to drag ratio are compared with its rigid counterpart. A single non-rotating NREL phase VI blade is studied here as a first step towards analyzing the concept for the rotating case. Compared with the rigid blade, the membrane blade has a higher slope of the lift curve. For higher angles of attack, lift and drag coefficients as well as the lift to drag ratio is higher for the membrane blade. A single non-rotating blade is studied here as a first step towards analyzing the concept for the rotating case.

  17. Interactions of Al(acac)3 with cell membranes and model phospholipid bilayers.

    PubMed

    Suwalsky, M; Ungerer, B; Villena, F; Norris, B; Cárdenas, H; Zatta, P

    1999-07-15

    Aluminum is a neurotoxic agent; however, little information has been obtained regarding its molecular cytotoxicity and the effects on the stability of biological membranes. This is mainly due to the ill-defined chemical speciation of the metal compounds. For this reason, the present study used aluminum acetylacetonate, (Al(acac)3), a neutral, chemically well-defined, hydrolytically stable and lipophilic compound. To understand the molecular mechanism of its interaction with cell membranes, Al(acac)3 was incubated with human erythrocytes, isolated toad skin and molecular models of biomembranes. The latter consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoyphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The results showed that Al(acac)3 interacted with the erythrocyte membrane modifying its normal discoid morphology to both echinocytic and stomatocytic shapes. This finding indicates that the Al complex was inserted in both the outer and inner layers of the red cell membrane, a conclusion supported by X-ray diffraction analyses of DMPC and DMPE bilayers. Electrophysiological measurements performed on toad skin revealed a significant decrease in the potential difference and short-circuit current responses after application of Al(acac)3, effects interpreted to reflect inhibition of the active transport of ions. Al(acac)3 was active on both surfaces of the skin suggesting that the membrane was permeated by the metal complex. It is concluded that Al(acac)3 both alters the molecular structure of the lipid bilayer, thereby modifying the biophysical properties of the cell membrane, and changes its physiological properties. PMID:10499289

  18. Interaction of divalent metal ions with human translocase of inner membrane of mitochondria Tim23.

    PubMed

    Feng, Wei; Zhang, Yongqiang; Deng, Honghua; Li, Shu Jie

    2016-06-17

    The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23IMS) and the interaction of presequence peptides with Tim23IMS in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23IMS. The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim23IMS is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex.

  19. Interaction of divalent metal ions with human translocase of inner membrane of mitochondria Tim23.

    PubMed

    Feng, Wei; Zhang, Yongqiang; Deng, Honghua; Li, Shu Jie

    2016-06-17

    The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23IMS) and the interaction of presequence peptides with Tim23IMS in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23IMS. The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim23IMS is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex. PMID:27178215

  20. Can Xanthophyll-Membrane Interactions Explain Their Selective Presence in the Retina and Brain?

    PubMed Central

    Widomska, Justyna; Zareba, Mariusz; Subczynski, Witold Karol

    2016-01-01

    Epidemiological studies demonstrate that a high dietary intake of carotenoids may offer protection against age-related macular degeneration, cancer and cardiovascular and neurodegenerative diseases. Humans cannot synthesize carotenoids and depend on their dietary intake. Major carotenoids that have been found in human plasma can be divided into two groups, carotenes (nonpolar molecules, such as β-carotene, α-carotene or lycopene) and xanthophylls (polar carotenoids that include an oxygen atom in their structure, such as lutein, zeaxanthin and β-cryptoxanthin). Only two dietary carotenoids, namely lutein and zeaxanthin (macular xanthophylls), are selectively accumulated in the human retina. A third carotenoid, meso-zeaxanthin, is formed directly in the human retina from lutein. Additionally, xanthophylls account for about 70% of total carotenoids in all brain regions. Some specific properties of these polar carotenoids must explain why they, among other available carotenoids, were selected during evolution to protect the retina and brain. It is also likely that the selective uptake and deposition of macular xanthophylls in the retina and brain are enhanced by specific xanthophyll-binding proteins. We hypothesize that the high membrane solubility and preferential transmembrane orientation of macular xanthophylls distinguish them from other dietary carotenoids, enhance their chemical and physical stability in retina and brain membranes and maximize their protective action in these organs. Most importantly, xanthophylls are selectively concentrated in the most vulnerable regions of lipid bilayer membranes enriched in polyunsaturated lipids. This localization is ideal if macular xanthophylls are to act as lipid-soluble antioxidants, which is the most accepted mechanism through which lutein and zeaxanthin protect neural tissue against degenerative diseases. PMID:27030822

  1. Proteoliposomes as Tool for Assaying Membrane Transporter Functions and Interactions with Xenobiotics

    PubMed Central

    Scalise, Mariafrancesca; Pochini, Lorena; Giangregorio, Nicola; Tonazzi, Annamaria; Indiveri, Cesare

    2013-01-01

    Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. Using recently developed reconstitution strategies, transporters can be inserted in artificial bilayers with the same orientation as in the cell membranes and in the absence of other interfering molecular systems. These methodologies are very suitable for studying kinetic parameters and molecular mechanisms. After the first applications on mitochondrial transporters, in the last decade, proteoliposomes obtained with optimized methodologies have been used for studying plasma membrane transporters and defining their functional and kinetic properties and structure/function relationships. A lot of information has been obtained which has clarified and completed the knowledge on several transporters among which the OCTN sub-family members, transporters for neutral amino acid, B0AT1 and ASCT2, and others. Transporters can mediate absorption of substrate-like derivatives or drugs, improving their bioavailability or can interact with these compounds or other xenobiotics, leading to side/toxic effects. Therefore, proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H2O2 and some drugs. Several mechanisms have been defined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially important tool in drug discovery. PMID:24300519

  2. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

    PubMed Central

    Thelin, William R.; Chen, Yun; Gentzsch, Martina; Kreda, Silvia M.; Sallee, Jennifer L.; Scarlett, Cameron O.; Borchers, Christoph H.; Jacobson, Ken; Stutts, M. Jackson; Milgram, Sharon L.

    2007-01-01

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation. PMID:17235394

  3. Preferential interactions in pigmented, polymer blends - C.I. Pigment Blue 15:4 and C.I. Pigment Red 122 - as used in a poly(carbonate)-poly(butylene terephthalate) polymer blend.

    PubMed

    Fagelman, K E; Guthrie, J T

    2005-11-18

    Some important characteristics of selected pigments have been evaluated, using the inverse gas chromatography (IGC) technique, that indicate the occurrence of preferential interactions in pigmented polymer blends. Attention has been given to copper phthalocyanine pigments and to quinacridone pigments incorporated in polycarbonate-poly(butylene terephthalate) blends. Selected supporting techniques were used to provide supplementary information concerning the pigments of interest, C.I. Pigment Blue 15:4 and C.I. Pigment Red 122. For C.I. Pigment Red 122 and for C.I. Pigment Blue, the dispersive component of the surface free energy decreases as the temperature increases, indicating the relative ease with which the molecules can be removed from the surface.

  4. Electrostatic interactions in phospholipid membranes revealed by coherent 2D IR spectroscopy

    PubMed Central

    Volkov, V. V.; Chelli, R.; Zhuang, W.; Nuti, F.; Takaoka, Y.; Papini, A. M.; Mukamel, S.; Righini, R.

    2007-01-01

    The inter- and intramolecular interactions of the carbonyl moieties at the polar interface of a phospholipid membrane are probed by using nonlinear femtosecond infrared spectroscopy. Two-dimensional IR correlation spectra separate homogeneous and inhomogeneous broadenings and show a distinct cross-peak pattern controlled by electrostatic interactions. The inter- and intramolecular electrostatic interactions determine the inhomogeneous character of the optical response. Using molecular dynamics simulation and the nonlinear exciton equations approach, we extract from the spectra short-range structural correlations between carbonyls at the interface. PMID:17881567

  5. Selective trans-membrane transport of alkali and alkaline earth cations through graphene oxide membranes based on cation-π interactions.

    PubMed

    Sun, Pengzhan; Zheng, Feng; Zhu, Miao; Song, Zhigong; Wang, Kunlin; Zhong, Minlin; Wu, Dehai; Little, Reginald B; Xu, Zhiping; Zhu, Hongwei

    2014-01-28

    Graphene and graphene oxide (G-O) have been demonstrated to be excellent filters for various gases and liquids, showing potential applications in areas such as molecular sieving and water desalination. In this paper, the selective trans-membrane transport properties of alkali and alkaline earth cations through a membrane composed of stacked and overlapped G-O sheets ("G-O membrane") are investigated. The thermodynamics of the ion transport process reveal that the competition between the generated thermal motions and the interactions of cations with the G-O sheets results in the different penetration behaviors to temperature variations for the considered cations (K(+), Mg(2+), Ca(2+), and Ba(2+)). The interactions between the studied metal atoms and graphene are quantified by first-principles calculations based on the plane-wave-basis-set density functional theory (DFT) approach. The mechanism of the selective ion trans-membrane transportation is discussed further and found to be consistent with the concept of cation-π interactions involved in biological systems. The balance between cation-π interactions of the cations considered with the sp(2) clusters of G-O membranes and the desolvation effect of the ions is responsible for the selectivity of G-O membranes toward the penetration of different ions. These results help us better understand the ion transport process through G-O membranes, from which the possibility of modeling the ion transport behavior of cellular membrane using G-O can be discussed further. The selectivity toward different ions also makes G-O membrane a promising candidate in areas of membrane separations.

  6. Preferential Remedies for Employment Discrimination

    ERIC Educational Resources Information Center

    Edwards, Harry T.; Zaretsky, Barry L.

    1975-01-01

    An overview of the problem of preferential remedies to achieve equal employment opportunities for women and minority groups. Contends that "color blindness" will not end discrimination but that some form of "color conscious" affirmative action program must be employed. Temporary preferential treatment is justified, according to the author, by the…

  7. Characterization of hybrid bilayer membranes on silver electrodes as biocompatible SERS substrates to study membrane-protein interactions.

    PubMed

    Millo, Diego; Bonifacio, Alois; Moncelli, Maria Rosa; Sergo, Valter; Gooijer, Cees; van der Zwan, Gert

    2010-11-01

    Hybrid bilayer lipid membranes (HBMs) were built on roughened silver electrodes exhibiting surface-enhanced Raman scattering (SERS) activity. The HBM consisted of a first layer of octadecanethiol (ODT) directly bound to the electrode surface, on which a second layer of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained by self-assembled phospholipid vesicle fusion. The electrochemical properties of the HBM were investigated in situ by cyclic voltammetry (CV), AC voltammetry and electrochemical impedance spectroscopy (EIS). The results indicate that our HBMs are well-formed, and their insulating properties are comparable to those observed for HBM supported by smooth metal substrates. The interaction between the bilayer and the human enzyme cytochrome P450 2D6 (CYP2D6) was investigated. Surface-enhanced resonance Raman scattering (SERRS) measurements in combination with AC and EIS, performed on the same electrode sample, proved that the CYP2D6 is immobilized on the HBM without evident alterations of its active site and without significant perturbations of the bilayer architecture. This study yields novel insights into the properties of HBMs built on roughened surfaces, providing in situ electrochemical characterization of a substrate which is suitable for studying peripheral membrane proteins with SERRS spectroscopy.

  8. Reacquisition of cocaine conditioned place preference and its inhibition by previous social interaction preferentially affect D1-medium spiny neurons in the accumbens corridor

    PubMed Central

    Prast, Janine M.; Schardl, Aurelia; Schwarzer, Christoph; Dechant, Georg; Saria, Alois; Zernig, Gerald

    2014-01-01

    We investigated if counterconditioning with dyadic (i.e., one-to-one) social interaction, a strong inhibitor of the subsequent reacquisition of cocaine conditioned place preference (CPP), differentially modulates the activity of the diverse brain regions oriented along a mediolateral corridor reaching from the interhemispheric sulcus to the anterior commissure, i.e., the nucleus of the vertical limb of the diagonal band, the medial septal nucleus, the major island of Calleja, the intermediate part of the lateral septal nucleus, and the medial accumbens shell and core. We also investigated the involvement of the lateral accumbens core and the dorsal caudate putamen. The anterior cingulate 1 (Cg1) region served as a negative control. Contrary to our expectations, we found that all regions of the accumbens corridor showed increased expression of the early growth response protein 1 (EGR1, Zif268) in rats 2 h after reacquisition of CPP for cocaine after a history of cocaine CPP acquisition and extinction. Previous counterconditioning with dyadic social interaction inhibited both the reacquisition of cocaine CPP and the activation of the whole accumbens corridor. EGR1 activation was predominantly found in dynorphin-labeled cells, i.e., presumably D1 receptor-expressing medium spiny neurons (D1-MSNs), with D2-MSNs (immunolabeled with an anti-DRD2 antibody) being less affected. Cholinergic interneurons or GABAergic interneurons positive for parvalbumin, neuropeptide Y or calretinin were not involved in these CPP-related EGR1 changes. Glial cells did not show any EGR1 expression either. The present findings could be of relevance for the therapy of impaired social interaction in substance use disorders, depression, psychosis, and autism spectrum disorders. PMID:25309368

  9. Cotton plasma membrane intrinsic protein 2s (PIP2s) selectively interact to regulate their water channel activities and are required for fibre development.

    PubMed

    Li, Deng-Di; Ruan, Xiang-Mei; Zhang, Jie; Wu, Ya-Jie; Wang, Xiu-Lan; Li, Xue-Bao

    2013-08-01

    Aquaporins are thought to be associated with water transport and play important roles in cotton (Gossypium hirsutum) fibre elongation. Among aquaporins, plasma membrane intrinsic proteins (PIPs) constitute a plasma-membrane-specific subfamily and are further subdivided into PIP1 and PIP2 groups. In this study, four fibre-preferential GhPIP2 genes were functionally characterized. The selective interactions among GhPIP2s and their interaction proteins were studied in detail to elucidate the molecular mechanism of cotton fibre development. GhPIP2;3 interacted with GhPIP2;4 and GhPIP2;6, but GhPIP2;6 did not interact with GhPIP2;4. Coexpression of GhPIP2;3/2;4 or GhPIP2;3/2;6 resulted in a positive cooperative effect which increased the permeability coefficient of oocytes, while GhPIP2;4/2;6 did not. GhBCP2 (a blue copper-binding protein) inhibited GhPIP2;6 water channel activity through their interaction. Overexpression of GhPIP2 genes in yeast induced longitudinal growth of the host cells. By contrast, knockdown of expression of GhPIP2 genes in cotton by RNA interference markedly hindered fibre elongation. In conclusion, GhPIP2 proteins are the primary aquaporin isoforms in fibres. They selectively form hetero-oligomers in order to regulate their activities to meet the requirements for rapid fibre elongation.

  10. The interaction of small molecules with phospholipid membranes studied by 1H NOESY NMR under magic-angle spinning.

    PubMed

    Scheidt, Holger A; Huster, Daniel

    2008-01-01

    The interaction of small molecules with lipid membranes and the exact knowledge of their binding site and bilayer distribution is of great pharmacological importance and represents an active field of current biophysical research. Over the last decade, a highly resolved 1H solid-state NMR method has been developed that allows measuring localization and distribution of small molecules in membranes. The classical solution 1H NMR NOESY technique is applied to lipid membrane samples under magic-angle spinning (MAS) and NOESY cross-relaxation rates are determined quantitatively. These rates are proportional to the contact probability between molecular segments and therefore an ideal tool to study intermolecular interactions in membranes. Here, we review recent 1H MAS NOESY applications that were carried out to study lateral lipid organization in mixed membranes and the interaction of membranes with water, ethanol, small aromatic compounds, peptides, fluorescence labels, and lipophilic nucleosides.

  11. Comparative X-ray studies on the interaction of carotenoids with a model phosphatidylcholine membrane.

    PubMed

    Suwalsky, Mario; Hidalgo, Paulina; Strzalka, Kazimierz; Kostecka-Gugala, Anna

    2002-01-01

    The interaction of structurally different carotenoids with a membrane molecular model was examined by X-ray diffraction. The selected compounds were beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin, and additionally carotane, a fully saturated derivative of beta-carotene. They present similarities and differences in their rigidity, the presence of terminal ionone rings and hydroxy and epoxy groups bound to the rings. The membrane models were multibilayers of dipalmitoylphosphatidylcholine (DPPC), chosen for this investigation because the 3 nm thickness of the hydrophobic core of its bilayer coincides with the thickness of the hydrophobic core of thylakoid membranes and the length of the carotenoid molecules. Results indicate that the six compounds induced different types and degrees of structural perturbations to DPPC bilayers in aqueous media. They were interpreted in terms of the molecular characteristics of DPPC and the carotenoids. Lycopene and violaxanthin induced the highest structural damage to the acyl chain and polar headgroup regions of DPPC bilayers, respectively. PMID:11926524

  12. Interaction of penicillin G with the human erythrocyte membrane and models.

    PubMed

    Suwalsky, M; Villena, F; Aguilar, F; Sotomayor, C P

    1996-01-01

    Penicillin G (PEN) is a widely used antibiotic whose mechanism of action is related to the interference with the synthesis of bacteria cell wall. In order to evaluate its perturbing effect upon human cell membranes PEN was made to interact with human erythrocytes, isolated resealed human erythrocyte membranes and molecular models. The latter were multibilayers of the phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) as well as DMPC large unilamellar vesicles. These studies were performed by scanning electron microscopy, fluorescence spectroscopy and X-ray diffraction methods. The observed results coincide in that PEN did not exert any significant effect upon the structures of the red cell membrane neither on its molecular models. This is in agreement with its reported lack of major toxicity and hematological reactions. PMID:8639231

  13. Calcium and protons affect the interaction of neurotransmitters and anesthetics with anionic lipid membranes.

    PubMed

    Pérez-Isidoro, Rosendo; Ruiz-Suárez, J C

    2016-09-01

    We study how zwitterionic and anionic biomembrane models interact with neurotransmitters (NTs) and anesthetics (ATs) in the presence of Ca(2+) and different pH conditions. As NTs we used acetylcholine (ACh), γ-aminobutyric acid (GABA), and l-glutamic acid (LGlu). As ATs, tetracaine (TC), and pentobarbital (PB) were employed. By using differential scanning calorimetry (DSC), we analyzed the changes such molecules produce in the thermal properties of the membranes. We found that calcium and pH play important roles in the interactions of NTs and ATs with the anionic lipid membranes. Changes in pH promote deprotonation of the phosphate groups in anionic phospholipids inducing electrostatic interactions between them and NTs; but if Ca(2+) ions are in the system, these act as bridges. Such interactions impact the physical properties of the membranes in a similar manner that anesthetics do. Beyond the usual biochemical approach, we claim that these effects should be taken into account to understand the excitatory-inhibitory orchestrated balance in the nervous system. PMID:27362370

  14. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    PubMed

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides. PMID:27003128

  15. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    PubMed

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

  16. Membrane Interactions of Phytochemicals as Their Molecular Mechanism Applicable to the Discovery of Drug Leads from Plants.

    PubMed

    Tsuchiya, Hironori

    2015-10-16

    In addition to interacting with functional proteins such as receptors, ion channels, and enzymes, a variety of drugs mechanistically act on membrane lipids to change the physicochemical properties of biomembranes as reported for anesthetic, adrenergic, cholinergic, non-steroidal anti-inflammatory, analgesic, antitumor, antiplatelet, antimicrobial, and antioxidant drugs. As well as these membrane-acting drugs, bioactive plant components, phytochemicals, with amphiphilic or hydrophobic structures, are presumed to interact with biological membranes and biomimetic membranes prepared with phospholipids and cholesterol, resulting in the modification of membrane fluidity, microviscosity, order, elasticity, and permeability with the potencies being consistent with their pharmacological effects. A novel mechanistic point of view of phytochemicals would lead to a better understanding of their bioactivities, an insight into their medicinal benefits, and a strategic implication for discovering drug leads from plants. This article reviews the membrane interactions of different classes of phytochemicals by highlighting their induced changes in membrane property. The phytochemicals to be reviewed include membrane-interactive flavonoids, terpenoids, stilbenoids, capsaicinoids, phloroglucinols, naphthodianthrones, organosulfur compounds, alkaloids, anthraquinonoids, ginsenosides, pentacyclic triterpene acids, and curcuminoids. The membrane interaction's applicability to the discovery of phytochemical drug leads is also discussed while referring to previous screening and isolating studies.

  17. Still embedded together binding to membranes regulates Bcl-2 protein interactions.

    PubMed

    Leber, B; Lin, J; Andrews, D W

    2010-09-23

    The dysregulation of apoptosis is a key step in developing tumours, and mediates resistance to cancer therapy. Many different signals for cell death converge on permeabilization of the outer mitochondrial membrane, which is controlled by the Bcl-2 family of proteins. The importance of this step is becoming increasingly relevant as the first generation of small molecules that inhibit the interaction of Bcl-2 family proteins enters clinical trials as anticancer agents. The Bcl-2 family can be divided into three classes: BH3-only proteins that are activated by various forms of cellular stress, Bax and Bak proteins that mediate mitochondrial membrane permeabilization, and inhibitory proteins such as Bcl-2 and Bcl-XL. The recently proposed embedded together model emphasizes the fact that many of the regulatory interactions between different classes of Bcl-2 family members occur at intracellular membranes, and binding to membranes causes conformational changes in the proteins that dictate functions in a dynamic manner. Within this context, recent results indicate that Bcl-XL functions as a dominant-negative Bax, a concept that resolves the paradox of similar structures but opposite functions of Bcl-XL and Bax. We have also shown that the conformational change that allows Bax to insert into the outer mitochondrial membrane is the rate-limiting step in the multistep process of Bax activation. Nevertheless, investigating the structure of activated Bax or Bak as monomers and as components of the oligomeric structures that mediate membrane permeabilization is the focus of ongoing research (and controversy) at many laboratories worldwide. PMID:20639903

  18. Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

    PubMed Central

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-01-01

    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), N-terminal 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. PMID:22044229

  19. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

    PubMed

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-01-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers. PMID:27363513

  20. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

    PubMed

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-07-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers.

  1. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy

    PubMed Central

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-01-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers. PMID:27363513

  2. Nature Utilizes Unusual High London Dispersion Interactions for Compact Membranes Composed of Molecular Ladders.

    PubMed

    Wagner, J Philipp; Schreiner, Peter R

    2014-03-11

    London dispersion interactions play a key role in nature, in particular, in membranes that constitute natural barriers. Here we demonstrate that the spatial alignment of "molecular ladders" ([n]ladderanes), i.e., highly unusual and strained all-trans-fused cyclobutane moieties, leads to much larger attractive dispersion interactions as compared to alkyl chains of the same length. This provides a rationale for the occurrence of peculiar ladderane fatty acids in the dense cell walls of anammox bacteria. Despite the energetic penalty paid for the assembly of such strained polycycles, the advantage lies in significantly higher, dispersion-dominated interaction energies as compared to straight-chain hydrocarbon moieties commonly found in fatty acids. We discern the dispersion contributions to the total interaction energies using a variety of computational methods including modern dispersion-corrected density functional theory and high level ab initio approaches. Utilizing larger assemblies, we also show that the intermolecular interactions behave additively.

  3. Cell Receptor-Basement Membrane Interactions in Health and Disease: a Kidney-Centric View

    PubMed Central

    Borza, Corina M.; Chen, Xiwu; Zent, Roy; Pozzi, Ambra

    2016-01-01

    Cell-extracellular matrix (ECM) interactions are essential for tissue development, homeostasis, and response to injury. Basement membranes (BMs) are specialized ECMs that separate epithelial or endothelial cells from stromal components and interact with cells via cellular receptors, including integrins and discoidin domain receptors. Disruption of cell-BM interactions due to either injury or genetic defects in either the ECM components or cellular receptors often lead to irreversible tissue injury and loss of organ function. Animal models that lack specific BM components or receptors either globally or in selective tissues have been used to help with our understanding of the molecular mechanisms whereby cell-BM interactions regulate organ function in physiological and pathological conditions. We review recently published work on animal models that explore how cell-BM interactions regulate kidney homeostasis in both health and disease. PMID:26610916

  4. Self-interaction chromatography as a tool for optimizing conditions for membrane protein crystallization.

    PubMed

    Gabrielsen, Mads; Nagy, Lisa A; DeLucas, Lawrence J; Cogdell, Richard J

    2010-01-01

    The second virial coefficient, or B value, is a measurement of how well a protein interacts with itself in solution. These interactions can lead to protein crystallization or precipitation, depending on their strength, with a narrow range of B values (the 'crystallization slot') being known to promote crystallization. A convenient method of determining the B value is by self-interaction chromatography. This paper describes how the light-harvesting complex 1-reaction centre core complex from Allochromatium vinosum yielded single straight-edged crystals after iterative cycles of self-interaction chromatography and crystallization. This process allowed the rapid screening of small molecules and detergents as crystallization additives. Here, a description is given of how self-interaction chromatography has been utilized to improve the crystallization conditions of a membrane protein.

  5. Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

    PubMed

    Kucharska, Iga; Liang, Binyong; Ursini, Nicholas; Tamm, Lukas K

    2016-09-13

    Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 μM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa. PMID:27532487

  6. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

  7. Comparisons of the interaction of propranolol and timolol with model and biological membrane systems

    SciTech Connect

    Herbette, L.; Katz, A.M.; Sturtevant, J.M.

    1983-09-01

    The nonspecific interaction of the beta-adrenergic blocking drugs, propranolol and timolol, with model and biological membranes has been investigated. Radioisotope measurements of the association of these drugs with dimyristoyl lecithin (DMPC) bilayers showed that both propranolol and timolol had a significantly greater molar association (mole of drug per mole of lipid) with DMPC above its phase transition temperature than below. Timolol had a much lower molar association with DMPC as compared with propranolol both above and below the phase transition temperature. For the DMPC model membrane system, the molar association of propranolol as measured by radioisotope and inferred from calorimetric studies was similar. Neutron diffraction utilizing propranolol deuterated in the naphthalene moiety showed that the naphthalene moiety of propranolol partitions into the hydrocarbon core of the DMPC lipid bilayer, and that the charged amine side chain is most likely positioned in the aqueous phospholipid head group region. For timolol, the association as measured by radioisotope methods was apparently greater than the partitioning inferred from calorimetric studies using freezing point depression analysis, suggesting a more complex interaction of timolol as compared with propranolol with the DMPC lipid bilayer. The association of propranolol with the SR membrane (mole of propranolol per mole of SR phospholipid) correlated with its ability to inhibit calcium uptake, whereas only a fraction of the total association of timolol with the SR membrane appeared to lead to inhibition of calcium uptake. Both propranolol and timolol appear to perturb the functional properties of the calcium pump protein in the SR membrane (inhibition of ATP-induced calcium uptake) indirectly by partitioning into the bulk lipid matrix of the SR lipid bilayer, although other sites of interaction cannot be excluded.

  8. New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

    PubMed

    Saavedra, Paula; Girona, Josefa; Bosquet, Alba; Guaita, Sandra; Canela, Núria; Aragonès, Gemma; Heras, Mercedes; Masana, Lluís

    2015-11-01

    Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.

  9. Interaction of the catalytic and the membrane subunits of an oxyanion-translocating ATPase.

    PubMed

    Dey, S; Dou, D; Tisa, L S; Rosen, B P

    1994-06-01

    Resistance to arsenical and antimonial compounds in Escherichia coli is due to active extrusion of these compounds from cells expressing the ars operon. The arsenical pump is an ion-translocating ATPase which consists of two polypeptide components, the ArsA and ArsB proteins. The ArsB protein, the inner membrane component of the pump, has been shown to function as the membrane anchor for the catalytic subunit, the ArsA protein. The properties and nature of interaction between these two components of the pump were investigated using an in vitro binding assay. Purified ArsA protein bound to the membrane in a saturable manner. In the absence of arsenite or antimonite an apparent positive cooperativity in the binding of the ArsA protein to membrane vesicles containing the ArsB protein was observed. In the presence of arsenite or antimonite binding became hyperbolic, with a 10-fold decrease in the concentration of ArsA protein required for half-maximal binding, without any change in the stoichiometry of the complex. Addition of ATP had little affect on membrane binding of the ArsA ATPase subunit. In the presence or absence of the anionic substrates binding was maximal in a pH range 7.5-8.5.

  10. Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry

    PubMed Central

    Smith, Rachel A. S.; Nabok, Aleksey; Blakeman, Ben J. F.; Xue, Wei-Feng; Abell, Benjamin; Smith, David P.

    2015-01-01

    There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity. PMID:26172440

  11. Model membrane interaction and DNA-binding of antimicrobial peptide Lasioglossin II derived from bee venom.

    PubMed

    Bandyopadhyay, Susmita; Lee, Meryl; Sivaraman, J; Chatterjee, Chiradip

    2013-01-01

    Lasioglossins, a new family of antimicrobial peptide, have been shown to have strong antimicrobial activity with low haemo-lytic and mast cell degranulation activity, and exhibit cytotoxic activity against various cancer cells in vitro. In order to understand the active conformation of these pentadecapeptides in membranes, we have studied the interaction of Lasioglossin II (LL-II), one of the members of Lasioglossins family with membrane mimetic micelle Dodecylphosphocholine (DPC) by fluorescence, Circular Dichroism (CD) and two dimensional (2D) (1)H NMR spectroscopy. Fluorescence experiments provide evidence of interaction of the N-terminal tryptophan residue of LL-II with the hydrophobic core of DPC micelle. CD results show an extended chain conformation of LL-II in water which is converted to a partial helical conformation in the presence of DPC micelle. Moreover we have determined the first three-dimensional NMR structure of LL-II bound to DPC micelle with rmsd of 0.36Å. The solution structure of LL-II shows hydrophobic and hydrophilic core formation in peptide pointing towards different direction in the presence of DPC. This amphipathic structure may allow this peptide to penetrate deeply into the interfacial region of negatively charged membranes and leading to local membrane destabilization. Further we have elucidated the DNA binding ability of LL-II by agarose gel retardation and fluorescence quenching experiments.

  12. The anticancer drug cytarabine does not interact with the human erythrocyte membrane.

    PubMed

    Suwalsky, Mario; Hernández, Pedro L; Villena, Fernando; Sotomayor, Carlos P

    2003-01-01

    Cytarabine, an analog of deoxycytidine, is an important agent in the treatment of ovarian carcinoma, acute myeloid and lymphoblastic leukemia. Its mechanism of action has been attributed to an interference with DNA replication. The plasma membrane has received increasing attention as a possible target of antitumor drugs, where the drugs may act as growth factor antagonists and receptor blockers, interfere with mitogenic signal transduction or exert direct cytotoxic effects. Furthermore, it has been reported that drugs that exert their antiproliferative effect by interacting with DNA generally cause structural and functional membrane alterations which may be essential for growth inhibition by these agents. This paper describes the studies undertaken to determine the structural effects induced by cytarabine to cell membranes. The results showed that cytarabine, at a concentration about one thousand times higher than that found in plasma when it is therapeutically administered, did not induce significant structural perturbation in any of these systems. Therefore, it can be unambiguously concluded that this widely used anticancer drug does not interact at all with erythrocyte membranes. PMID:14713170

  13. Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry.

    PubMed

    Smith, Rachel A S; Nabok, Aleksey; Blakeman, Ben J F; Xue, Wei-Feng; Abell, Benjamin; Smith, David P

    2015-01-01

    There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity.

  14. [Interaction of Membrane and Calcium Oscillators in Cardiac Pacemaker Cells: Mathematical Modeling].

    PubMed

    Ryvkin, A M; Zorin, N M; Moskvin, A S; Solovyova, O E; Markhasin, V S

    2015-01-01

    An integrative model of the calcium dynamics in cardiac pacemaker cells is developed taking into account a synergetic effect of the interaction between an outer membrane oscillator and an intracellular calcium oscillator ("membrane and Ca(2+)-clock"). The main feature of the model is a description of the stochastic dynamics of Ca2+ release units within the electron-conformational mechanism of the functioning of ryanodine-sensitive calcium channels. It is shown that interaction of two cellular oscillators provides a stable action potential generation in the cardiac pacemaker cells even in the case of the stochastic Ca2+ dynamics. We studied in detail the effect of ryanodine channels sensitivity to an increase in the intracellular calcium concentration in sarcoplasmic reticulum and in the dyadic space on the behavior of calcium-release system. A parametric analysis of the integrative model of pacemaker cells is performed. PMID:26841508

  15. Interaction of elastocapillary flows in parallel microchannels across a thin membrane

    NASA Astrophysics Data System (ADS)

    Reddy, S. P.; Samy, R. A.; Sen, A. K.

    2016-10-01

    We report the interaction of counter elastocapillary flows in parallel microchannels across a thin membrane. At the crossing point, the interaction between the capillary flows via the thin membrane leads to significant retardation of capillary flow. The drop in velocity at the crossing point and velocity variation after the crossing point are predicted using the analytical model and measured from experiments. A non-dimensional parameter J, which is the ratio of the capillary force to the mechanical restoring force, governs the drop in velocity at the crossing point with the maximum drop of about 60% for J = 1. The meniscus velocity after the crossing point decreases (J < 0.5), remains constant (0.5 < J < 0.6), or increases (J > 0.6) depending on the value of J. The proposed technique can be applied for the manipulation of capillary flows in microchannels.

  16. Harvesting energy of interaction between bacteria and bacteriophage in a membrane-less fuel cell.

    PubMed

    Gupta, Ragini; Bekele, Wasihun; Ghatak, Animangsu

    2013-11-01

    When a fuel and oxidant flow in laminar contact through a micro-fluidic channel, a sharp interface appears between the two liquids, which eliminate the need of a proton exchange membrane. This principle has been used to generate potential in a membrane-less fuel cell. This study use such a cell to harvest energy of interaction between a bacteria having negative charge on its surface and a bacteriophage with positive and negative charges on its tail and head, respectively. When Klebsiella pneumoniae (Kp6) and phage (P-Kp6) are pumped through a fuel cell fitted with two copper electrodes placed at its two sides, interaction between these two charged species at the interface results in a constant open circuit potential which varies with concentration of charged species but gets generated for both specific and non-specific bacteria and phage system. Oxygenation of bacteria or phage however diminishes the potential unlike in conventional microbial fuel cells.

  17. Interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) with cell and model membranes.

    PubMed

    Suwalsky, M; Benites, M; Villena, F; Aguilar, F; Sotomayor, C P

    1996-12-01

    2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a component of the "agent orange' whose toxicity has been extensively studied without definite conclusions. In order to evaluate its perturbing effect upon cell membranes, 2,4-D was made to interact with human erythrocytes and molecular models. These studies were performed by scanning electron microscopy on red cells, fluorescence spectroscopy on dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles and X-ray diffraction on multilayers of DMPC and dimyristoylphosphatidylethanolamine (DMPE). It was observed that 2,4-D induced a pronounced shape change to the erythrocytes. This effect is explained by the herbicide interaction with the outer monolayer of the red cell membrane. PMID:8972711

  18. Spin-label studies on rat liver and heart plasma membranes: do probe-probe interactions interfere with the measurement of membrane properties?

    PubMed

    Sauerheber, R D; Gordon, L M; Crosland, R D; Kuwahara, M D

    1977-02-24

    The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the "expanded" spectra were significantly more precise than those obtained from the "unexpanded" spectra. Both procedures were used to study the effects of various I(12,3) probe concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following: The polarity-corrected order parameters of liver (31 degrees) and heart (22 degrees) membranes were found to be independent of the probe concentration, if experimentally-determined low I(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that "intrinsic" membrane properties may be measured at these low probe/lipid ratios. Here, "intrinsic" properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label. At higher I(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the "apparent" fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to "low" probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of "liquid-lines" (or "fluid components") in the unexpanded ESR spectra was a convenient marker of high probe concentrations. A thermotropic phase

  19. Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes.

    PubMed

    Agre, P; Virshup, D; Bennett, V

    1984-09-01

    Two new vascular smooth muscle relaxants, bepridil and cetiedil, were found to possess specific CaM-inhibitory properties which resembled those of trifluoperazine. Trifluoperazine, bepridil, and cetiedil inhibited Ca2+-dependent 125I-CaM binding to erythrocyte membranes and CaM activation of membrane Ca2+-ATPase with IC50 values of approximately 12, approximately 17, and approximately 40 microM, respectively. This does not appear to be the result of a nonspecific hydrophobic interaction since inhibition was not observed with micromolar concentrations of many other hydrophobic agents. The predominant inhibition of binding and Ca2+-ATPase activation was competitive with respect to CaM. Bepridil and cetiedil bind directly to CaM since these drugs displaced [3H]trifluoperazine from sites on CaM. Inhibition of Ca2+-ATPase and binding by the drugs was not due to interference with the catalytic activity of this enzyme since: (a) neither inhibition of CaM-independent basal Ca2+-ATPase activity nor inhibition of proteolytically-activated Ca2+-ATPase activities were produced by these agents, and (b) no drug-induced inhibition of CaM binding was detected when membranes were preincubated with these agents but washed prior to addition of 125I-CaM. Thus, bepridil and cetiedil competitively inhibit Ca2+-dependent interactions of CaM with erythrocyte membranes, most likely by a direct interaction between these drugs and CaM. The principal clinical actions of these drugs may be explained by their interactions with CaM or CaM-related proteins leading to reduced activation of Ca2+-regulated enzymes in certain other tissues, such as myosin light chain kinase in vascular smooth muscle.

  20. Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane.

    PubMed

    Witschas, Katja; Jobin, Marie-Lise; Korkut, Dursun Nizam; Vladan, Maria Magdalena; Salgado, Gilmar; Lecomte, Sophie; Vlachova, Viktorie; Alves, Isabel D

    2015-05-01

    The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific.

  1. Biomechanics and thermodynamics of nanoparticle interactions with plasma and endosomal membrane lipids in cellular uptake and endosomal escape.

    PubMed

    Peetla, Chiranjeevi; Jin, Shihua; Weimer, Jonathan; Elegbede, Adekunle; Labhasetwar, Vinod

    2014-07-01

    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(D,L-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  2. Fluorescent microscope system to monitor real-time interactions between focused ultrasound, echogenic drug delivery vehicles, and live cell membranes.

    PubMed

    Ibsen, Stuart; Benchimol, Michael; Esener, Sadik

    2013-01-01

    Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5MPa the membranes were shown to completely fragment while at intensities below 1MPa the membranes pop open and slowly unfold. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery.

  3. Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity.

    PubMed

    Vogel, Alexander; Nikolaus, Jörg; Weise, Katrin; Triola, Gemma; Waldmann, Herbert; Winter, Roland; Herrmann, Andreas; Huster, Daniel

    2014-07-01

    Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance--PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol--using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

  4. Protein corona mitigates the cytotoxicity of graphene oxide by reducing its physical interaction with cell membrane.

    PubMed

    Duan, Guangxin; Kang, Seung-gu; Tian, Xin; Garate, Jose Antonio; Zhao, Lin; Ge, Cuicui; Zhou, Ruhong

    2015-10-01

    Many recent studies have shown that the way nanoparticles interact with cells and biological molecules can vary greatly in the serum-containing or serum-free culture medium. However, the underlying molecular mechanisms of how the so-called "protein corona" formed in serum medium affects nanoparticles' biological responses are still largely unresolved. Thus, it is critical to understand how absorbed proteins on the surfaces of nanoparticles alter their biological effects. In this work, we have demonstrated with both experimental and theoretical approaches that protein BSA coating can mitigate the cytotoxicity of graphene oxide (GO) by reducing its cell membrane penetration. Our cell viability and cellular uptake experiments showed that protein corona decreased cellular uptake of GO, thus significantly mitigating the potential cytotoxicity of GO. The electron microscopy images also confirmed that protein corona reduced the cellular morphological damage by limiting GO penetration into the cell membrane. Further molecular dynamics (MD) simulations validated the experimental results and revealed that the adsorbed BSA in effect weakened the interaction between the phospholipids and graphene surface due to a reduction of the available surface area plus an unfavorable steric effect, thus significantly reducing the graphene penetration and lipid bilayer damaging. These findings provide new insights into the underlying molecular mechanism of this important graphene protein corona interaction with cell membranes, and should have implications in future development of graphene-based biomedical applications.

  5. Energy of the interaction between membrane lipid domains calculated from splay and tilt deformations

    NASA Astrophysics Data System (ADS)

    Galimzyanov, T. R.; Molotkovsky, R. J.; Kheyfets, B. B.; Akimov, S. A.

    2013-01-01

    Specific domains, called rafts, are formed in cell membranes. Similar lipid domains can be formed in model membranes as a result of phase separation with raft size may remaining small (˜10-100 nm) for a long time. The characteristic lifetime of a nanoraft ensemble strongly depends on the nature of mutual raft interactions. The interaction energy between the boundaries of two rafts has been calculated under the assumption that the thickness of the raft bilayer is greater than that of the surrounding membrane, and elastic deformations appear in order to smooth the thickness mismatch at the boundary. When rafts approach each other, deformations from their boundaries overlap, making interaction energy profile sophisticated. It has been shown that raft merger occurs in two stages: rafts first merge in one monolayer of the lipid bilayer and then in another monolayer. Each merger stage requires overcoming of an energy barrier of about 0.08-0.12 k BT per 1 nm of boundary length. These results allow us to explain the stability of the ensemble of finite sized rafts.

  6. Rapid mobilization of membrane lipids in wheat leaf sheaths during incompatible interactions with Hessian fly.

    PubMed

    Zhu, Lieceng; Liu, Xuming; Wang, Haiyan; Khajuria, Chitvan; Reese, John C; Whitworth, R Jeff; Welti, Ruth; Chen, Ming-Shun

    2012-07-01

    Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time polymerase chain reaction analyses of 168 lipid-metabolism-related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack.

  7. The importance of hydrogen bonding in sphingomyelin's membrane interactions with co-lipids.

    PubMed

    Slotte, J Peter

    2016-02-01

    Sphingomyelin is an important constituent of mammalian cell membranes. Its molecular structure is N-acyl-D-erythro-sphingosylphosphorylcholine. The N-acyls in sphingomyelin often contain 16-24 carbons that are mostly saturated chains; however, the monounsaturated 24:1(Δ15c) acyl chain is also common. In addition to the more saturated nature of sphingomyelins, compared to physiologically relevant glycerophospholipids, also their hydrogen bonding properties are very different from the glycerophospholipids. Sphingomyelins form extensive intramolecular hydrogen bonds (from the 3OH of the long-chain base to phosphate oxygens of the head group), but also intermolecular hydrogen bonding involving the NH of the long-chain base are important for sphingomyelin (and sphingolipid) properties in membrane environments. Hydrogen bonding involving sphingomyelin has been shown to markedly stabilize interactions with both cholesterol and ceramide in fully hydrated bilayers. Such interactions contribute to the propensity of saturated sphingomyelin to form a liquid-ordered phase together with cholesterol, or a gel phase with saturated ceramides. The purpose of this review is to present recent experimental and computational evidence in support of the importance of hydrogen bonding for the interaction of sphingomyelin with other membrane lipids.

  8. Effects of cooperative ion-channel interactions on the dynamics of excitable membranes

    NASA Astrophysics Data System (ADS)

    Zarubin, Dmitry; Zhuchkova, Ekaterina; Schreiber, Susanne

    2012-06-01

    Cooperative interactions between ion channels are known to exist, but have so far received relatively little attention in the study of excitable membranes. Based on bifurcation analysis and stochastic simulations of an extended Morris-Lecar model, we show that cooperativity and anticooperativity can modify the range of sustained firing and cell-intrinsic noise, induce multistability, and account for a number of experimental observations, including prolongation of action-potential duration. We hypothesize that channel interactions could be an efficient mechanism to regulate the activity of neurons or cardiac muscle cells.

  9. Valosin-containing protein-interacting membrane protein (VIMP) links the endoplasmic reticulum with microtubules in concert with cytoskeleton-linking membrane protein (CLIMP)-63.

    PubMed

    Noda, Chikano; Kimura, Hana; Arasaki, Kohei; Matsushita, Mitsuru; Yamamoto, Akitsugu; Wakana, Yuichi; Inoue, Hiroki; Tagaya, Mitsuo

    2014-08-29

    The distribution and morphology of the endoplasmic reticulum (ER) in mammalian cells depend on both dynamic and static interactions of ER membrane proteins with microtubules (MTs). Cytoskeleton-linking membrane protein (CLIMP)-63 is exclusively localized in sheet-like ER membranes, typical structures of the rough ER, and plays a pivotal role in the static interaction with MTs. Our previous study showed that the 42-kDa ER-residing form of syntaxin 5 (Syn5L) regulates ER structure through the interactions with both CLIMP-63 and MTs. Here, we extend our previous study and show that the valosin-containing protein/p97-interacting membrane protein (VIMP)/SelS is also a member of the family of proteins that shape the ER by interacting with MTs. Depletion of VIMP causes the spreading of the ER to the cell periphery and affects an MT-dependent process on the ER. Although VIMP can interact with CLIMP-63 and Syn5L, it does not interact with MT-binding ER proteins (such as Reep1) that shape the tubular smooth ER, suggesting that different sets of MT-binding ER proteins are used to organize different ER subdomains.

  10. Retention of pesticide Endosulfan by nanofiltration: influence of organic matter-pesticide complexation and solute-membrane interactions.

    PubMed

    De Munari, Annalisa; Semiao, Andrea Joana Correia; Antizar-Ladislao, Blanca

    2013-06-15

    Nanofiltration (NF) is a well-established process used in drinking water production to effectively remove Natural Organic Matter (NOM) and organic micropollutants. The presence of NOM has been shown to have contrasting results on micropollutant retention by NF membranes and removal mechanisms are to date poorly understood. The permeate water quality can therefore vary during operation and its decrease would be an undesired outcome for potable water treatment. It is hence important to establish the mechanisms involved in the removal of organic micropollutants by NF membranes in the presence of NOM. In this study, the retention mechanisms of pesticide Endosulfan (ES) in the presence of humic acids (HA) by two NF membranes, TFC-SR2 and TFC-SR3, a "loose" and a "tight" membrane, respectively, were elucidated. The results showed that two mechanisms were involved: (1) the formation of ES-HA complexes (solute-solute interactions), determined from solid-phase micro-extraction (SPME), increased ES retention, and (2) the interactions between HA and the membrane (solute-membrane interactions) increased membrane molecular weight cut-off (MWCO) and decreased ES retention. HA concentration, pH, and the ratio between micropollutant molecular weight (MW) and membrane MWCO were shown to influence ES retention mechanisms. In the absence of HA-membrane interactions at pH 4, an increase of HA concentration increased ES retention from 60% to 80% for the TFC-SR2 and from 80% to 95% for the TFC-SR3 due to ES-HA complex formation. At pH 8, interactions between HA and the loose TFC-SR2 increased the membrane MWCO from 460 to 496 g/mol and ES retention decreased from 55% to 30%, as HA-membrane interactions were the dominant mechanism for ES retention. In contrast, for the "tight" TFC-SR3 membrane the increase in the MWCO (from 165 to 179 g/mol), was not sufficient to decrease ES retention which was dominated by ES-HA interactions. Quantification of the contribution of both solute

  11. Role of Cell Membrane-Vector Interactions in Successful Gene Delivery.

    PubMed

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2016-08-16

    Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex

  12. Role of Cell Membrane-Vector Interactions in Successful Gene Delivery.

    PubMed

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2016-08-16

    Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex

  13. Computational analysis of the tether-pulling experiment to probe plasma membrane-cytoskeleton interaction in cells

    NASA Astrophysics Data System (ADS)

    Schumacher, Kristopher R.; Popel, Aleksander S.; Anvari, Bahman; Brownell, William E.; Spector, Alexander A.

    2009-10-01

    Tethers are thin membrane tubes that can be formed when relatively small and localized forces are applied to cellular membranes and lipid bilayers. Tether pulling experiments have been used to better understand the fine membrane properties. These include the interaction between the plasma membrane and the underlying cytoskeleton, which is an important factor affecting membrane mechanics. We use a computational method aimed at the interpretation and design of tether pulling experiments in cells with a strong membrane-cytoskeleton attachment. In our model, we take into account the detailed information in the topology of bonds connecting the plasma membrane and the cytoskeleton. We compute the force-dependent piecewise membrane deflection and bending as well as modes of stored energy in three major regions of the system: body of the tether, membrane-cytoskeleton attachment zone, and the transition zone between the two. We apply our method to three cells: cochlear outer hair cells (OHCs), human embryonic kidney (HEK) cells, and Chinese hamster ovary (CHO) cells. OHCs have a special system of pillars connecting the membrane and the cytoskeleton, and HEK and CHO cells have the membrane-cytoskeleton adhesion arrangement via bonds (e.g., PIP2), which is common to many other cells. We also present a validation of our model by using experimental data on CHO and HEK cells. The proposed method can be an effective tool in the analyses of experiments to probe the properties of cellular membranes.

  14. Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR.

    PubMed

    Bodor, Andrea; Kövér, Katalin E; Mäler, Lena

    2015-03-01

    Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used 31P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and 31P T2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T2 relaxation are observed at higher temperatures. A comparison of 31P T1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules--in particular for the negatively charged DMPG--while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide-membrane interactions. PMID:25497765

  15. Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

    PubMed

    Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

    2014-08-01

    With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells.

  16. The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.

    PubMed

    Zhang, Shao-Qing; Kulp, Daniel W; Schramm, Chaim A; Mravic, Marco; Samish, Ilan; DeGrado, William F

    2015-03-01

    α Helices are a basic unit of protein secondary structure and therefore the interaction between helices is crucial to understanding tertiary and higher-order folds. Comparing subtle variations in the structural and sequence motifs between membrane and soluble proteins sheds light on the different constraints faced by each environment and elucidates the complex puzzle of membrane protein folding. Here, we demonstrate that membrane and water-soluble helix pairs share a small number of similar folds with various interhelical distances. The composition of the residues that pack at the interface between corresponding motifs shows that hydrophobic residues tend to be more enriched in the water-soluble class of structures and small residues in the transmembrane class. The latter group facilitates packing via sidechain- and backbone-mediated hydrogen bonds within the low-dielectric membrane milieu. The helix-helix interactome space, with its associated sequence preferences and accompanying hydrogen-bonding patterns, should be useful for engineering, prediction, and design of protein structure.

  17. Interaction of TonB with the Outer Membrane Receptor FpvA of Pseudomonas aeruginosa

    PubMed Central

    Adams, Hendrik; Zeder-Lutz, Gabrielle; Schalk, Isabelle; Pattus, Franc; Celia, Hervé

    2006-01-01

    Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonBpp) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonBpp was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonBpp interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro. PMID:16885443

  18. The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.

    PubMed

    Zhang, Shao-Qing; Kulp, Daniel W; Schramm, Chaim A; Mravic, Marco; Samish, Ilan; DeGrado, William F

    2015-03-01

    α Helices are a basic unit of protein secondary structure and therefore the interaction between helices is crucial to understanding tertiary and higher-order folds. Comparing subtle variations in the structural and sequence motifs between membrane and soluble proteins sheds light on the different constraints faced by each environment and elucidates the complex puzzle of membrane protein folding. Here, we demonstrate that membrane and water-soluble helix pairs share a small number of similar folds with various interhelical distances. The composition of the residues that pack at the interface between corresponding motifs shows that hydrophobic residues tend to be more enriched in the water-soluble class of structures and small residues in the transmembrane class. The latter group facilitates packing via sidechain- and backbone-mediated hydrogen bonds within the low-dielectric membrane milieu. The helix-helix interactome space, with its associated sequence preferences and accompanying hydrogen-bonding patterns, should be useful for engineering, prediction, and design of protein structure. PMID:25703378

  19. Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains.

    PubMed Central

    Kalus, Ina; Hodel, Alois; Koch, Annett; Kleene, Ralf; Edwardson, J Michael; Schrader, Michael

    2002-01-01

    Syncollin, a novel pancreatic zymogen granule protein, is present on the luminal side of the granule membrane. To address the function of syncollin, we searched for putative binding partners. Cross-linking experiments with purified syncollin, and granule content and membrane proteins revealed a direct interaction between syncollin and GP-2, a major glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. An interaction was also observed when cross-linking was performed with recombinant GP-2. In addition, syncollin could be cross-linked to itself, supporting the suggestion that it exists as a homo-oligomer. Cleavage of the GPI anchor of GP-2 by treatment of granule membranes with phosphatidylinositol-specific phospholipase C had no effect on the membrane attachment of syncollin, indicating that it is not mediated exclusively via an interaction with GP-2. Syncollin was found to be associated with detergent-insoluble cholesterol/glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose-density gradients and also contained GP-2, the lectin ZG16p, sulphated matrix proteoglycans and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) syntaxin 3 and synaptobrevin 2. Our results indicate that membrane-associated syncollin is a component of lipid rafts, where it interacts both with GP-2 and membrane lipids. We suggest that the syncollin-GP-2 complex might play a role in signal transduction across the granule membrane. PMID:11853552

  20. Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction*

    PubMed Central

    Inder, Kerry L.; Lau, Chiyan; Loo, Dorothy; Chaudhary, Natasha; Goodall, Andrew; Martin, Sally; Jones, Alun; van der Hoeven, Dharini; Parton, Robert G.; Hill, Michelle M.; Hancock, John F.

    2009-01-01

    The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3. PMID:19661056

  1. Interactions of Borneol with DPPC Phospholipid Membranes: A Molecular Dynamics Simulation Study

    PubMed Central

    Yin, Qianqian; Shi, Xinyuan; Ding, Haiou; Dai, Xingxing; Wan, Guang; Qiao, Yanjiang

    2014-01-01

    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. PMID:25383679

  2. Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes

    PubMed Central

    Jelokhani-Niaraki, Masoud; Hodges, Robert S.; Meissner, Joseph E.; Hassenstein, Una E.; Wheaton, Laura

    2008-01-01

    To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 ≥ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis. PMID:18621820

  3. Interactions of borneol with DPPC phospholipid membranes: a molecular dynamics simulation study.

    PubMed

    Yin, Qianqian; Shi, Xinyuan; Ding, Haiou; Dai, Xingxing; Wan, Guang; Qiao, Yanjiang

    2014-11-06

    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.

  4. Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.

    PubMed Central

    Sigal, C T; Zhou, W; Buser, C A; McLaughlin, S; Resh, M D

    1994-01-01

    Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation. Images PMID:7527558

  5. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

    PubMed

    Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-11-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at

  6. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

    PubMed

    Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-11-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at

  7. A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding.

    PubMed

    Khan, Hanif M; He, Tao; Fuglebakk, Edvin; Grauffel, Cédric; Yang, Boqian; Roberts, Mary F; Gershenson, Anne; Reuter, Nathalie

    2016-03-29

    Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought. PMID:27028646

  8. Effects of structure on the interactions between five natural antimicrobial compounds and phospholipids of bacterial cell membrane on model monolayers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be...

  9. Interactions of Pleckstrin Homology Domains with Membranes: Adding Back the Bilayer via High-Throughput Molecular Dynamics.

    PubMed

    Yamamoto, Eiji; Kalli, Antreas C; Yasuoka, Kenji; Sansom, Mark S P

    2016-08-01

    A molecular simulation pipeline for determining the mode of interaction of pleckstrin homology (PH) domains with phosphatidylinositol phosphate (PIP)-containing lipid bilayers is presented. We evaluate our methodology for the GRP1 PH domain via comparison with structural and biophysical data. Coarse-grained simulations yield a 2D density landscape for PH/membrane interactions alongside residue contact profiles. Predictions of the membrane localization and interactions of 13 PH domains reveal canonical, non-canonical, and dual PIP-binding sites on the proteins. Thus, the PH domains associate with the PIP molecules in the membrane via a highly positively charged loop. Some PH domains exhibit modes of interaction with PIP-containing membranes additional to this canonical binding mode. All 13 PH domains cause a degree of local clustering of PIP molecules upon binding to the membrane. This provides a global picture of PH domain interactions with membranes. The high-throughput approach could be extended to other families of peripheral membrane proteins. PMID:27427480

  10. Fouling of nanofiltration, reverse osmosis, and ultrafiltration membranes by protein mixtures: the role of inter-foulant-species interaction.

    PubMed

    Wang, Yi-Ning; Tang, Chuyang Y

    2011-08-01

    Protein fouling of nanofiltration (NF), reverse osmosis (RO), and ultrafiltration (UF) membranes by bovine serum albumin (BSA), lysozyme (LYS), and their mixture was investigated under cross-flow conditions. The effect of solution chemistry, membrane properties, and permeate flux level was systematically studied. When the solution pH was within the isoelectric points (IEPs) of the two proteins (i.e., pH 4.7-10.4), the mixed protein system experienced more severe flux decline compared to the respective single protein systems, which may be attributed to the electrostatic attraction between the negatively charged BSA and positively charged LYS molecules. Unlike a typical single protein system, membrane fouling by BSA-LYS mixture was only weakly dependent on solution pH within this pH range, and increased ionic strength was found to enhance the membrane flux as a result of the suppressed BSA-LYS electrostatic attraction. Membrane fouling was likely controlled by foulant-fouled-membrane interaction under severe fouling conditions (elevated flux level and unfavorable solution chemistry that promotes fouling), whereas it was likely dominated by foulant-clean-membrane interaction under mild fouling conditions. Compared to nonporous NF and RO membranes, the porous UF membrane was more susceptible to dramatic flux decline due to the increased risk of membrane pore plugging. This study reveals that membrane fouling by mixed macromolecules may behave very differently from that by typical single foulant system, especially when the inter-foulant-species interaction dominates over the intra-species interaction in the mixed foulant system.

  11. Interaction of Phenylalanine with DPPC Model Membranes: More Than a Hydrophobic Interaction.

    PubMed

    Rosa, A S; Cutro, A C; Frías, M A; Disalvo, E A

    2015-12-31

    The negative free energy previously reported is explained by the stabilization of a PC-Phe (phosphocholine-phenylalanine) complex in the presence of water shown by the decrease in the symmetric stretching frequency of the phosphate group of the lipid (PO2(-)). An entropic contribution due to the disruption of the water network around the phenyl and in the membrane defect may be invoked. The dipole potential decrease is explained by the orientation of the carboxylate opposing to the CO of the lipids with oxygen moiety toward the low hydrated hydrocarbon core. The symmetric bending frequency of NH3(+) group of Phe, decreases in 5.2 cm(-1) in relation to water congruent with zeta potential shift to positive values. The Phe to DPPC dissociation constant is Kd = 2.23 ± 0.09 mM, from which the free energy change is about -4.54 kcal/mol at 25 °C. This may be due to hydrophobic contributions and two hydrogen bonds.

  12. Synthesis, structural characterization, modal membrane interaction and anti-tumor cell line studies of nitrophenyl ferrocenes

    NASA Astrophysics Data System (ADS)

    Altaf, Ataf Ali; Lal, Bhajan; Badshah, Amin; Usman, Muhammad; Chatterjee, Pabitra B.; Huq, Fazlul; Ullah, Shafiq; Crans, Debbie C.

    2016-06-01

    A series of nitrophenyl ferrocens (A1 - A5) were synthesized and fully characterized in solid state (using CHN analysis, FTIR and single crystal XRD) as well as in solution phase (1H &13C NMR and UV-visible spectroscopy). Micelle interface interactions of these compounds were explored and found to have ability across a micelle membrane interface. Interestingly, these compounds exhibited π-electronic push pull systems and oxidation of ferrocene to ferrocenium on crossing the negative interface of the micelle membrane. Selective compounds were screened for antitumor activity against parental and drug resistant human ovarian tumor models i.e. A2780 and A2780cisR, A2780ZD0473R. Screened compounds were found to overcome resistance factor compared to cisplatin.

  13. The Role of the Plasma Membrane H+-ATPase in Plant–Microbe Interactions

    PubMed Central

    Elmore, James Mitch; Coaker, Gitta

    2011-01-01

    Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant–pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility. PMID:21300757

  14. Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes.

    PubMed

    Haxholm, Gitte W; Nikolajsen, Louise F; Olsen, Johan G; Fredsted, Jacob; Larsen, Flemming H; Goffin, Vincent; Pedersen, Stine F; Brooks, Andrew J; Waters, Michael J; Kragelund, Birthe B

    2015-06-15

    Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

  15. Interaction between the Ca2(+)-ATPase and the proteolipid in artificial membranes.

    PubMed

    Jóna, I; Martonosi, A

    1989-01-01

    The interaction between the Ca2+ transport ATPase and the proteolipid of rabbit sarcoplasmic reticulum was analyzed by fluorescence energy transfer, using the following donor: acceptor combinations: Ca2(+)-ATPase tryptophan----IAEDANS-proteolipid; IAEDANS-ATPase----IAF-proteolipid; IAEDANS-proteolipid----IAF-ATPase. The observed energy transfer may indicate weak interaction between the Ca2(+)-ATPase and proteolipid, but collisional energy transfer definitely contributes. The energy transfer was abolished by deoxycholate or sodium dodecylsulfate at concentrations sufficient to solubilize the membrane. In view of the low proteolipid content of sarcoplasmic reticulum and the weak interaction suggested by the energy transfer, at best only a small fraction of ATPase molecules could exist in the form of ATPase-proteolipid complexes.

  16. Effects of Naturally Occuring Arginine 14 Deletion on Phospholamban Conformational Dynamics and Membrane Interactions

    PubMed Central

    Vostrikov, Vitaly V.; Soller, Kailey J.; Ha, Kim N.; Gopinath, T.; Veglia, Gianluigi

    2014-01-01

    Phospholamban (PLN) is a single-pass membrane protein that regulates the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Phosphorylation of PLN at Ser16 reverses its inhibitory function under β-adrenergic stimulation, augmenting Ca2+ uptake in the sarcoplasmic reticulum and muscle contractility. PLN exists in two conformations; a T state, where the cytoplasmic domain is helical and absorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies from our group have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR techniques to compare the structural topology and conformational dynamics of monomeric PLN (PLNAFA) with that of the PLNR14del, a naturally occurring deletion mutant that is linked to the progression of dilated cardiomyopathy. We found that the behavior of the inhibitory transmembrane domain of PLNR14del is similar to that of the native sequence. In contrast, the conformational dynamics of R14del both in micelles and lipid membranes are enhanced. We conclude that the deletion of Arg14 in the cytoplasmic region weakens the interactions with the membrane and shifts the conformational equilibrium of PLN toward the disordered R state. This conformational transition is correlated with the loss-of-function character of this mutant and is corroborated by SERCA’s activity assays. These findings further support our hypothesis that SERCA function is fine-tuned by PLN conformational dynamics and begin to explain the aberrant regulation of SERCA by the R14del mutant. PMID:25251363

  17. Specific interactions of sticholysin I with model membranes: an NMR study.

    PubMed

    Castrillo, Inés; Araujo, Nelson A; Alegre-Cebollada, Jorge; Gavilanes, José G; Martínez-del-Pozo, Alvaro; Bruix, Marta

    2010-06-01

    Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N-terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by (1)H-NMR the environment of these regions in water and in the presence of membrane-mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1-30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix-turn-helix motif with the last alpha-helical segment matching the native helix-alpha(1) (residues 14-24) present in the complete protein. The first helix (residues 4-9) is less populated and is not present in the water-soluble protein structure. The characterization of wild-type StnI structure in micelles shows that the helix-alpha(1) is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp-111, Tyr-112, Trp-115, Tyr-132, Tyr-136, and Tyr-137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins.

  18. Specific interactions of sticholysin I with model membranes: an NMR study.

    PubMed

    Castrillo, Inés; Araujo, Nelson A; Alegre-Cebollada, Jorge; Gavilanes, José G; Martínez-del-Pozo, Alvaro; Bruix, Marta

    2010-06-01

    Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N-terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by (1)H-NMR the environment of these regions in water and in the presence of membrane-mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1-30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix-turn-helix motif with the last alpha-helical segment matching the native helix-alpha(1) (residues 14-24) present in the complete protein. The first helix (residues 4-9) is less populated and is not present in the water-soluble protein structure. The characterization of wild-type StnI structure in micelles shows that the helix-alpha(1) is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp-111, Tyr-112, Trp-115, Tyr-132, Tyr-136, and Tyr-137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins. PMID:20408172

  19. Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane.

    PubMed

    Derde, Melanie; Nau, Françoise; Guérin-Dubiard, Catherine; Lechevalier, Valérie; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique

    2015-04-01

    Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.

  20. Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

    SciTech Connect

    Parry, G.; Beck, J.C.; Moss, L.; Bartley, J. ); Ojakian, G.K. )

    1990-06-01

    The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. The authors have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembranous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. They suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS0O while colchicine and acrylamide did not. They hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

  1. The nucleolar protein Nop19p interacts preferentially with Utp25p and Dhr2p and is essential for the production of the 40S ribosomal subunit in Saccharomyces cerevisiae.

    PubMed

    Choque, Elodie; Marcellin, Marlène; Burlet-Schiltz, Odile; Gadal, Olivier; Dez, Christophe

    2011-01-01

    In eukaryotes, ribosome biogenesis is a process of major interest that requires more than 200 factors acting coordinately in time and space. Using genetic and proteomic studies, most of the components have now been identified. Based on its nucleolar localization, we characterized the protein encoded by the open reading frame YGR251W, we renamed Nop19p as playing an essential role in ribosome biogenesis. Depletion of the Nop19p in yeast impairs pre-rRNA processing at sites A₀, A₁ and A₂, leading to a strong decrease in 18S rRNA and 40S subunit levels. Nop19p is a component of 90S preribosomes which assembly is believed to result from stepwise incorporation of UTP modules. We show that Nop19p depletion does not impair the incorporation of UTP subcomplexes on preribosomes and conversely that depletion of UTP subcomplexes does not affect Nop19p recruitment on 90S preribosomes. TAP experiments under stringent conditions revealed that Nop19p interacts preferentially with the DEAH-box RNA helicase Dhr2p and Utp25p, both required for A 0, A 1 and A 2 cleavages. Nop19p appeared essential for the incorporation of Utp25p in preribosomes. In addition, our results suggest that in absence of Nop19p, Dhr2p remains trapped within aberrant preribosomes.

  2. The nucleolar protein Nop19p interacts preferentially with Utp25p and Dhr2p and is essential for the production of the 40S ribosomal subunit in Saccharomyces cerevisiae

    PubMed Central

    Choque, Elodie; Marcellin, Marlène; Burlet-Schiltz, Odile

    2011-01-01

    In eukaryotes, ribosome biogenesis is a process of major interest that requires more than 200 factors acting coordinately in time and space. Using genetic and proteomic studies, most of the components have now been identified. Based on its nucleolar localization, we characterized the protein encoded by the open reading frame YGR251W, we renamed Nop19p as playing an essential role in ribosome biogenesis. Depletion of the Nop19p in yeast impairs pre-rRNA processing at sites A0, A1 and A2, leading to a strong decrease in 18S rRNA and 40S subunit levels. Nop19p is a component of 90S preribosomes which assembly is believed to result from stepwise incorporation of UTP modules. We show that Nop19p depletion does not impair the incorporation of UTP subcomplexes on preribosomes and conversely that depletion of UTP subcomplexes does not affect Nop19p recruitment on 90S preribosomes. TAP experiments under stringent conditions revealed that Nop19p interacts preferentially with the DEAH-box RNA helicase Dhr2p and Utp25p, both required for A0, A1 and A2 cleavages. Nop19p appeared essential for the incorporation of Utp25p in preribosomes. In addition, our results suggest that in absence of Nop19p, Dhr2p remains trapped within aberrant preribosomes. PMID:21941128

  3. Concentration polarization of interacting solute particles in cross-flow membrane filtration

    SciTech Connect

    Bhattacharjee, S.; Kim, A.S.; Elimelech, M.

    1999-04-01

    A theoretical approach for predicting the influence of interparticle interactions on concentration polarization and the ensuing permeate flux decline during cross-flow membrane filtration of charged solute particles is presented. The Ornstein-Zernike integral equation is solved using appropriate closures corresponding to hard-spherical and long-range solute-solute interactions to predict the radial distribution function of the solute particles in a concentrated solution (dispersion). Two properties of the solution, namely the osmotic pressure and the diffusion coefficient, are determined on the basis of the radial distribution function at different solute concentrations. Incorporation of the concentration dependence of these two properties in the concentration polarization model comprising the convective-diffusion equation and the osmotic-pressure governed permeate flux equation leads to the coupled prediction of the solute concentration profile and the local permeate flux. The approach leads to a direct quantitative incorporation of solute-solute interactions in the framework of a standard theory of concentration polarization. The developed model is used to study the effects of ionic strength and electrostatic potential on the variations of solute diffusivity and osmotic pressure. Finally, the combined influence of these two properties on the permeate flux decline behavior during cross-flow membrane filtration of charged solute particles is predicted.

  4. Headgroup-dependent membrane catalysis of apelin-receptor interactions is likely.

    PubMed

    Langelaan, David N; Rainey, Jan K

    2009-07-30

    Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelin-APJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6-K12 is highly structured, with R6-L9 directly interacting with headgroups of the micelle. Type I beta-turns are initiated between R6 and L9, and a well-defined type IV beta-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptide-receptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced. PMID:19708686

  5. Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

    PubMed

    Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

    2011-09-15

    The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

  6. Electrodynamics of lipid membrane interactions in the presence of zwitterionic buffers.

    PubMed

    Koerner, Megan M; Palacio, Luis A; Wright, Johnnie W; Schweitzer, Kelly S; Ray, Bruce D; Petrache, Horia I

    2011-07-20

    Due to thermal motion and molecular polarizability, electrical interactions in biological systems have a dynamic character. Zwitterions are dipolar molecules that typically are highly polarizable and exhibit both a positive and a negative charge depending on the pH of the solution. We use multilamellar structures of common lipids to identify and quantify the effects of zwitterionic buffers that go beyond the control of pH. We use the fact that the repeat spacing of multilamellar lipid bilayers is a sensitive and accurate indicator of the force balance between membranes. We show that common buffers can in fact charge up neutral membranes. However, this electrostatic effect is not immediately recognized because of the concomitant modification of dispersion (van der Waals) forces. We show that although surface charging can be weak, electrostatic forces are significant even at large distances because of reduced ionic screening and reduced van der Waals attraction. The zwitterionic interactions that we identify are expected to be relevant for interfacial biological processes involving lipid bilayers, and for a wide range of biomaterials, including amino acids, detergents, and pharmaceutical drugs. An appreciation of zwitterionic electrodynamic character can lead to a better understanding of molecular interactions in biological systems and in soft materials in general.

  7. Mechanistic aspects of peptide-membrane interactions determined by optical, dielectric and piezoelectric techniques: an overview.

    PubMed

    Oliveira, Maria D L; Franco, Octavio L; Nascimento, Jessica M; de Melo, Celso P; Andrade, Cesar A S

    2013-11-01

    Antimicrobial peptides (AMPs) have been isolated from a wide variety of organisms that include microorganisms, plants, insects, frogs and mammals. As part of the innate immune system expressed in many tissues, AMPs are able to provide protection against invasion of foreign microorganisms and exhibit a broad spectrum of activity against bacteria, fungi and/or virus. Non-AMPs cell-penetrating peptides have been used as carriers for overcoming the membrane barrier and helping in the delivery of various molecules into the cell. Physicochemical peptide-lipid interactions studies can provide us with reliable molecular information about microbe defense response, including the elucidation of the prevailing mechanisms of its action, such as the barrel-stave, toroidal pore, carpet and detergent-like models. In this paper, we present an overview of the peptide-lipid mechanisms of interaction as well as discuss alternative techniques that could help to elucidate the peptides functionality. Quartz crystal microbalance (QCM), surface plasmon resonance (SPR) spectroscopy and electrochemical impedance spectroscopy (EIS) are useful techniques to investigate in details of the peptide-membrane interaction. The techniques here discussed could also offer specific and low-cost methods that can to shed some light over the different modes of action of AMPs, contributing to the development of drugs against infectious diseases.

  8. Interaction of antiaggregant molecule ajoene with membranes. An ESR and 1H, 2H, 31P-NMR study.

    PubMed

    Debouzy, J C; Neumann, J M; Hervé, M; Daveloose, D; Viret, J; Apitz-Castro, R

    1989-01-01

    The structure of ajoene, a molecule extracted from garlic, has been studied by 1H-NMR and its interaction with model membranes by 1H-, 2H-, 31-P-NMR and ESR experiments. This study clearly shows that the ajoene molecule is located deep in the layer and is close to the interlayer medium. Moreover while NMR experiments show that the membrane structure is only slightly affected by the presence of ajoene, ESR experiments reveal significant modifications in phospholipid dynamics. This interaction, observed before with the phenothiazine derivative, promazine, results in an increase of the membrane fluidity in its hydrophobic part and could be related to clinical properties of ajoene.

  9. Thermodynamic descriptors, profiles and driving forces in membrane receptor-ligand interactions.

    PubMed

    Pliska, Vladimir

    2010-12-01

    Extension of the (isothermal) Gibbs-Helmholtz equation for the heat capacity terms (ΔC(p)) allows formulating a temperature function of the free (Gibbs) energy change (ΔG). An approximation of the virtually unknown ΔC(p) temperature function enables then to determine and numerically solve temperature functions of thermodynamic parameters ΔH and ΔS (enthalpy and entropy change, respectively). Analytical solutions and respective numeric procedures for several such approximation formulas are suggested in the presented paper. Agreement between results obtained by this analysis with direct microcalorimetric measurements of ΔH (and ΔC(p) derived from them) was approved on selected cases of biochemical interactions presented in the literature. Analysis of several ligand-membrane receptor systems indicates that temperature profiles of ΔH and ΔS are parallel, largely not monotonic, and frequently attain both positive and negative values within the current temperature range of biochemical reactions. Their course is determined by the reaction change of heat capacity: temperature extremes (maximum or minimum) of both ΔH and ΔS occur at ΔC(p)=0, for most of these systems at roughly 285-305 K. Thus, the driving forces of these interactions may change from enthalpy-, entropy-, or enthalpy-entropy-driven in a narrow temperature interval. In contrast, thermodynamic parameters of ligand-macromolecule interactions in solutions (not bound to a membrane) mostly display a monotonic course. In the case of membrane receptors, thermodynamic discrimination between pharmacologically defined groups-agonists, partial agonists, antagonists-is in general not specified and can be achieved, in the best, solely within single receptor groups.

  10. Protein-ligand and membrane-ligand interactions in pharmacology: the case of the translocator protein (TSPO).

    PubMed

    Hatty, Claire R; Banati, Richard B

    2015-10-01

    The targets of many small molecule drugs are membrane proteins, and traditionally the focus of pharmacology is on the interaction between such receptors and their small molecule drug ligands. However, the lipid membranes of cells and organelles are increasingly appreciated as diverse and dynamic structures that also specifically interact with small molecule drugs and peptides, causing profound changes in the properties of these membranes, and modulating the function of the membrane and the proteins within it. Drug-membrane interactions are likely to have a role in both the therapeutic and toxic activity of a variety of compounds, and their role in the overall pharmacological effect of a drug needs to be understood more clearly. This is the case for the 18 kDa translocator protein (TSPO) and its ligands, where functions that were established based on pharmacological studies are being called into question. Re-examining the putative functions of the TSPO and the effects of its ligands reveals a need to consider in more detail the interplay between protein-ligand and membrane-ligand interactions, and the modulatory relationship between TSPO and the lipid membrane.

  11. Effect of pre-strain and excess length on unsteady fluid-structure interactions of membrane airfoils

    NASA Astrophysics Data System (ADS)

    Rojratsirikul, P.; Wang, Z.; Gursul, I.

    2010-04-01

    Aerodynamic characteristics of two-dimensional membrane airfoils were experimentally investigated in a wind tunnel. The effects of the membrane pre-strain and excess length on the unsteady aspects of the fluid-structure interaction were studied. The deformation of the membrane as a function of angle of attack and free-stream velocity was measured using a high-speed camera. These measurements were complemented by the measurements of unsteady velocity field with a high frame-rate Particle Image Velocimetry (PIV) system as well as smoke visualization. Membrane airfoils with excess length exhibit higher vibration modes, earlier roll-up of vortices, and smaller separated flow regions, whereas the membranes with pre-strain generally behave more similarly to a rigid airfoil. Measured frequencies of the membrane vibrations suggest a possible coupling with the wake instabilities at high incidences for all airfoils.

  12. Quantification of chemical mixture interactions modulating dermal absorption using a multiple membrane fiber array.

    PubMed

    Baynes, Ronald E; Xia, Xin Rui; Imran, Mudassar; Riviere, Jim E

    2008-03-01

    Dermal exposures to chemical mixtures can potentially increase or decrease systemic bioavailability of toxicants in the mixture. Changes in dermal permeability can be attributed to changes in physicochemical interactions between the mixture, the skin, and the solute of interest. These physicochemical interactions can be described as changes in system coefficients associated with molecular descriptors described by Abraham's linear solvation energy relationship (LSER). This study evaluated the effects of chemical mixtures containing either a solvent (ethanol) or a surfactant (sodium lauryl sulfate, SLS) on solute permeability and partitioning by quantifying changes in system coefficients in skin and a three-membrane-coated fiber (MCF) system, respectively. Regression analysis demonstrated that changes in system coefficients in skin were strongly correlated ( R2 = 0.89-0.98) to changes in system coefficients in the three-membrane MCF array with mixtures containing either 1% SLS or 50% ethanol. The PDMS fiber appeared to play a significant role (R2 = 0.84-0.85) in the MCF array in predicting changes in solute permeability, while the WAX fiber appeared to contribute less (R2 = 0.59-0.77) to the array than the other two fibers. On the basis of changes in system coefficients that are part of a LSER, these experiments were able to link physicochemical interactions in the MCF with those interactions in skin when either system is exposed to 1% SLS or 50% ethanol. These experiments further demonstrated the utility of a MCF array to adequately predict changes in dermal permeability when skin is exposed to mixtures containing either a surfactant or a solvent and provide some insight into the nature of the physiochemical interactions that modulate dermal absorptions.

  13. Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion

    PubMed Central

    1996-01-01

    Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of

  14. Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery.

    PubMed

    Patching, Simon G

    2014-01-01

    Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

  15. FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments

    PubMed Central

    Pincet, Frédéric; Adrien, Vladimir; Yang, Rong; Delacotte, Jérôme; Rothman, James E.; Urbach, Wladimir

    2016-01-01

    Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A. PMID:27387979

  16. FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.

    PubMed

    Pincet, Frédéric; Adrien, Vladimir; Yang, Rong; Delacotte, Jérôme; Rothman, James E; Urbach, Wladimir; Tareste, David

    2016-01-01

    Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A. PMID:27387979

  17. The interaction of the carbon nanoparticles with human cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

    2013-09-01

    The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

  18. The Interactions of Proinsulin with Insulin Receptors on the Plasma Membrane of the Liver

    PubMed Central

    Freychet, Pierre

    1974-01-01

    The interactions of proinsulin with the insulin-specific receptors were investigated in purified rat liver plasma membranes. These studies were designed to characterize the binding of proinsulin to the insulin receptors, to search for proinsulin-specific receptor sites, and to examine the possibility of proinsulin conversion at the insulin receptor site. Proinsulin was only 3-5% as potent as insulin in binding to insulin receptors. Proinsulin reacted with all of the insulin-specific receptors, and direct binding studies of [125I]porcine proinsulin and [125I]rat proinsulin did not reveal proinsulin-specific receptor sites other than the insulin receptors in rat liver membranes. Quantitative data derived from steady-state and transient-state comparative binding studies of both [125I]proinsulin and [125I]insulin indicated that a 20-fold lower association rate constant essentially accounts for the reduced affinity of proinsulin for the insulin receptors. The possibility of proinsulin conversion at the insulin receptor sites was investigated. Material recovered from the membranes upon dissociation of the proinsulin-receptor complex was intact proinsulin and did not exhibit any conversion by a variety of analytical methods. These results indicate that the lower affinity of proinsulin for the insulin receptor in the liver is an intrinsic property of the proinsulin molecule. The lower uptake of proinsulin by the insulin receptor represents, in addition to a slower degradation of the prohormone, a further mechanism by which proinsulin exerts prolonged, albeit reduced, action in vivo. PMID:4421396

  19. Effects of lipids on the interaction of SecA with model membranes.

    PubMed

    Ahn, T; Kim, J S; Lee, B C; Yun, C H

    2001-11-01

    The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus. Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1). When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E. coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E. coli PE. In addition, when the PE of E. coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40%. The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E. coli PE/DOG > E. coli PE > DOPC. These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes.

  20. Interactions of sarin with polyelectrolyte membranes: a molecular dynamics simulation study.

    PubMed

    Lee, Ming-Tsung; Vishnyakov, Aleksey; Gor, Gennady Yu; Neimark, Alexander V

    2013-01-10

    Nanostructured polyelectrolyte membranes (PEMs), which are widely used as permselective diffusion barriers in fuel cell technologies and electrochemical processing, are considered as protective membranes suitable for blocking warfare toxins, including water-soluble nerve agents such as sarin. In this article, we examine the mechanisms of sorption and diffusion of sarin in hydrated PEMs by means of atomistic molecular dynamics simulations. Three PEMs are considered: Nafion, sulfonated polystyrene (sPS) that forms the hydrophilic subphase of segregated sPS-polyolefin block copolymers, and random sPS-polyethylene copolymer. We found that sarin concentrates at the interface between the hydrophilic and hydrophobic subphases of hydrated Nafion acting as a surfactant. In hydrated sPS, where the scale of water-polymer segregation is much smaller (1-2 nm), sarin also interacts favorably with hydrophobic and hydrophilic components. Water diffusion slows as the sarin content increases despite the overall increase in solvent content, which suggests that sarin and water have somewhat different pathways through the segregated membrane. Upon replacement of counterions of monovalent potassium with those of divalent calcium, sarin diffusion slows but remains substantial in all ionomers considered, especially at high sarin concentrations. The behavior of sarin is similar to that of its common simulant, dimethyl methylphosphonate. PMID:23205740

  1. Satellite observed preferential states in soil moisture

    NASA Astrophysics Data System (ADS)

    Vilasa, Luis U.; De Jeu, Richard A. M.; Dolman, Han A. J.; Wang, Guojie

    2013-04-01

    This study presents observational evidence for the existence of preferential states in soil moisture content. Recently there has been much debate about the existence, location and explanations for preferential states in soil moisture. A number of studies have provided evidence either in support or against the hypothesis of a positive feedback mechanism between soil moisture and subsequent precipitation in certain regions. Researchers who support the hypothesis that preferential states in soil moisture holds information about land atmosphere feedback base their theory on the impact of soil moisture on the evaporation process. Evaporation recycles moisture to the atmosphere and soil moisture has a direct impact on the supply part of this process but also on the partitioning of the available energy for evaporation. According to this theory, the existence of soil moisture bimodality can be used as an indication of possible land-atmosphere feedbacks, to be compared with model simulations of soil moisture feedbacks. On the other hand, other researchers argue that seasonality in the meteorological conditions in combination with the non-linearity of soil moisture response alone can induce bimodality. In this study we estimate the soil moisture bimodality at a global scale as derived from the recently available 30+ year ESA Climate Change Initative satellite soil moisture dataset. An Expectation-Maximization iterative algorithm is used to find the best Gaussian Mixture Model, pursuing the highest likelihood for soil moisture bimodality. With this approach we mapped the regions where bi-modal probability distribution of soil moisture appears for each month for the period between 1979-2010. These bimodality areas are analyzed and compared to maps of model simulations of soil moisture feedbacks. The areas where more than one preferential state exists compare surprisingly well with the map of land-atmosphere coupling strength from model simulations. This approach might

  2. Application of a membrane model to the sorptive interactions of humic substances.

    PubMed Central

    Wershaw, R L

    1989-01-01

    Humic substances, the dark-colored, natural organic polyelectrolytes that are found in practically all soils, sediments, and natural water, strongly interact with both inorganic and organic pollutants. Inorganic cationic species generally undergo complexation reactions with humic substances. The binding of cations, such as cupric ions, by humic substances often markedly reduces their toxicity to aquatic organisms. Some inorganic anionic species, in the presence of metal ions, are sorbed by humic substances. In these instances the metal ions appear to form bridges between the humic substances and the anions. Several different types of interactions take place between organic compounds and humic materials. Hydrophobic organic species partition into either insoluble or soluble humic substances. The insoluble humic substances will remove hydrophobic organic compounds from the aqueous phase, thereby rendering them less mobile. However, soluble humic substances will solubilize hydrophobic organics, increasing their mobility. Other types of interactions between humic substances and organic compounds, such as adsorption and ion exchange, also have been observed. These various interactions between humic substances and pollutants are important in governing their fate and movement in natural water systems, and, for this reason, a detailed understanding of the mechanisms of the interaction is important. A recently developed membrane model of the structure of humic substances is described; this model enables one to better understand the physical-chemical properties of these materials. Images FIGURE 2. FIGURE 3. PMID:2533555

  3. PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein

    PubMed Central

    Khelashvili, George; Saunders, Christine; Erreger, Kevin; Javitch, Jonathan A.; Sitte, Harald H.; Weinstein, Harel; Matthies, Heinrich J.G.; Galli, Aurelio

    2014-01-01

    Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms. PMID:24880859

  4. Disease mutations in desmoplakin inhibit Cx43 membrane targeting mediated by desmoplakin–EB1 interactions

    PubMed Central

    Patel, Dipal M.; Dubash, Adi D.; Kreitzer, Geri

    2014-01-01

    Mechanisms by which microtubule plus ends interact with regions of cell–cell contact during tissue development and morphogenesis are not fully understood. We characterize a previously unreported interaction between the microtubule binding protein end-binding 1 (EB1) and the desmosomal protein desmoplakin (DP), and demonstrate that DP–EB1 interactions enable DP to modify microtubule organization and dynamics near sites of cell–cell contact. EB1 interacts with a region of the DP N terminus containing a hotspot for pathogenic mutations associated with arrhythmogenic cardiomyopathy (AC). We show that a subset of AC mutations, in addition to a mutation associated with skin fragility/woolly hair syndrome, impair gap junction localization and function by misregulating DP–EB1 interactions and altering microtubule dynamics. This work identifies a novel function for a desmosomal protein in regulating microtubules that affect membrane targeting of gap junction components, and elucidates a mechanism by which DP mutations may contribute to the development of cardiac and cutaneous diseases. PMID:25225338

  5. Interactions of dendritic glycopolymer with erythrocytes, red blood cell ghosts and membrane enzymes.

    PubMed

    Wrobel, Dominika; Janaszewska, Anna; Appelhans, Dietmar; Voit, Brigitte; Bryszewska, Maria; Maly, Jan

    2015-12-30

    Interactions between maltose functionalized hyperbranched poly(ethylene imine)s (95% maltose decoration denoted as Mal-PEI A; 33% maltose decoration denoted as Mal-PEI B) and red blood cells (RBCs) and between red blood cell membranes were investigated. We monitored the degree of hemolysis, the change in cell shape, the influence of polymers on the fluidity of the cell membrane and some cell membrane enzymes to determine their possible cytotoxic impact on them. To observe the extent of hemolysis, the RBCs were incubated with different concentrations of Mal-PEIs. The first significant lysis of RBCs was observed after 6h of incubation. Prolongation of the incubation time increased the number of ruptured cells. Moreover, we observed that Mal-PEI B was more hemolytic than Mal-PEI A in buffer solution. In contrast, an incubation of RBCs with Mal-PEIs in human plasma significantly decreased the hemolytic process and showed higher hemolytic property of Mal-PEI A compared to Mal-PEI B. Also several changes in the shape of the RBCs occurred after incubation with Mal-PEIs. Some of the erythrocytes shrank (echinocytes), but their morphology generally remained unchanged during the incubation. As shown by fluorescence experiments, both polymers induced the increase of fluidity of RBCs membranes. In summary, both types of hyperbranched poly(ethylene imine)s were practically non-hemolytic even at high polymer concentrations. Mal-PEI B was slightly more noxious than the Mal-PEI A in a buffer solution, while in blood plasma, the situation was opposite. Decrease of Na+/K+ ATPase and total ATPase enzymes activity was related with molecule size and number of maltose groups on the surface of molecule. The low hemolytic properties only observed at higher concentration (100μM and 400μM) indicated that Mal-PEIs are promising macromolecules in the area of drug delivery systems. PMID:26498371

  6. Interactions of dendritic glycopolymer with erythrocytes, red blood cell ghosts and membrane enzymes.

    PubMed

    Wrobel, Dominika; Janaszewska, Anna; Appelhans, Dietmar; Voit, Brigitte; Bryszewska, Maria; Maly, Jan

    2015-12-30

    Interactions between maltose functionalized hyperbranched poly(ethylene imine)s (95% maltose decoration denoted as Mal-PEI A; 33% maltose decoration denoted as Mal-PEI B) and red blood cells (RBCs) and between red blood cell membranes were investigated. We monitored the degree of hemolysis, the change in cell shape, the influence of polymers on the fluidity of the cell membrane and some cell membrane enzymes to determine their possible cytotoxic impact on them. To observe the extent of hemolysis, the RBCs were incubated with different concentrations of Mal-PEIs. The first significant lysis of RBCs was observed after 6h of incubation. Prolongation of the incubation time increased the number of ruptured cells. Moreover, we observed that Mal-PEI B was more hemolytic than Mal-PEI A in buffer solution. In contrast, an incubation of RBCs with Mal-PEIs in human plasma significantly decreased the hemolytic process and showed higher hemolytic property of Mal-PEI A compared to Mal-PEI B. Also several changes in the shape of the RBCs occurred after incubation with Mal-PEIs. Some of the erythrocytes shrank (echinocytes), but their morphology generally remained unchanged during the incubation. As shown by fluorescence experiments, both polymers induced the increase of fluidity of RBCs membranes. In summary, both types of hyperbranched poly(ethylene imine)s were practically non-hemolytic even at high polymer concentrations. Mal-PEI B was slightly more noxious than the Mal-PEI A in a buffer solution, while in blood plasma, the situation was opposite. Decrease of Na+/K+ ATPase and total ATPase enzymes activity was related with molecule size and number of maltose groups on the surface of molecule. The low hemolytic properties only observed at higher concentration (100μM and 400μM) indicated that Mal-PEIs are promising macromolecules in the area of drug delivery systems.

  7. Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb.

    PubMed

    Patti, Monica; Fenollar-Ferrer, Cristina; Werner, Andreas; Forrest, Lucy R; Forster, Ian C

    2016-09-01

    Voltage-dependence of Na(+)-coupled phosphate cotransporters of the SLC34 family arises from displacement of charges intrinsic to the protein and the binding/release of one Na(+) ion in response to changes in the transmembrane electric field. Candidate coordination residues for the cation at the Na1 site were previously predicted by structural modeling using the x-ray structure of dicarboxylate transporter VcINDY as template and confirmed by functional studies. Mutations at Na1 resulted in altered steady-state and presteady-state characteristics that should be mirrored in the conformational changes induced by membrane potential changes. To test this hypothesis by functional analysis, double mutants of the flounder SLC34A2 protein were constructed that contain one of the Na1-site perturbing mutations together with a substituted cysteine for fluorophore labeling, as expressed in Xenopus oocytes. The locations of the mutations were mapped onto a homology model of the flounder protein. The effects of the mutagenesis were characterized by steady-state, presteady-state, and fluorometric assays. Changes in fluorescence intensity (ΔF) in response to membrane potential steps were resolved at three previously identified positions. These fluorescence data corroborated the altered presteady-state kinetics upon perturbation of Na1, and furthermore indicated concomitant changes in the microenvironment of the respective fluorophores, as evidenced by changes in the voltage dependence and time course of ΔF. Moreover, iodide quenching experiments indicated that the aqueous nature of the fluorophore microenvironment depended on the membrane potential. These findings provide compelling evidence that membrane potential and cation interactions induce significant large-scale structural rearrangements of the protein. PMID:27602725

  8. Design of Amphiphilic Protein Maquettes: Controlling Assembly, Membrane Insertion, and Cofactor Interactions

    SciTech Connect

    Discher,B.; Noy, D.; Strzalka, J.; Ye, S.; Moser, C.; Lear, J.; Blasie, K.; Dutton, L.

    2005-01-01

    We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) {alpha}-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-{alpha}-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-{alpha}-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-{alpha}-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b{sub H} heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc{sub 1}. These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.

  9. Viral potassium channels as a robust model system for studies of membrane-protein interaction.

    PubMed

    Braun, Christian J; Lachnit, Christine; Becker, Patrick; Henkes, Leonhard M; Arrigoni, Cristina; Kast, Stefan M; Moroni, Anna; Thiel, Gerhard; Schroeder, Indra

    2014-04-01

    The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking. PMID:23791706

  10. Interaction of Bile Salts with Model Membranes Mimicking the Gastrointestinal Epithelium: A Study by Isothermal Titration Calorimetry.

    PubMed

    Coreta-Gomes, Filipe M; Martins, Patrícia A T; Velazquez-Campoy, Adrián; Vaz, Winchil L C; Geraldes, Carlos F G; Moreno, Maria João

    2015-08-25

    Bile salts (BS) are biosurfactants synthesized in the liver and secreted into the intestinal lumen where they solubilize cholesterol and other hydrophobic compounds facilitating their gastrointestinal absorption. Partition of BS toward biomembranes is an important step in both processes. Depending on the loading of the secreted BS micelles with endogeneous cholesterol and on the amount of cholesterol from diet, this may lead to the excretion or absorption of cholesterol, from cholesterol-saturated membranes in the liver or to gastrointestinal membranes, respectively. The partition of BS toward the gastrointestinal membranes may also affect the barrier properties of those membranes affecting the permeability for hydrophobic and amphiphilic compounds. Two important parameters in the interaction of the distinct BS with biomembranes are their partition coefficient and the rate of diffusion through the membrane. Altogether, they allow the calculation of BS local concentrations in the membrane as well as their asymmetry in both membrane leaflets. The local concentration and, most importantly, its asymmetric distribution in the bilayer are a measure of induced membrane perturbation, which is expected to significantly affect its properties as a cholesterol donor and hydrophobic barrier. In this work we have characterized the partition of several BS, nonconjugated and conjugated with glycine, to large unilamellar vesicles (LUVs) in the liquid-disordered phase and with liquid-ordered/liquid-disordered phase coexistence, using isothermal titration calorimetry (ITC). The partition into the liquid-disordered bilayer was characterized by large partition coefficients and favored by enthalpy, while association with the more ordered membrane was weak and driven only by the hydrophobic effect. The trihydroxy BS partitions less efficiently toward the membranes but shows faster translocation rates, in agreement with a membrane protective effect of those BS. The rate of translocation

  11. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    PubMed

    Rydberg, Hanna A; Kunze, Angelika; Carlsson, Nils; Altgärde, Noomi; Svedhem, Sofia; Nordén, Bengt

    2014-07-01

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  12. Interaction of the Antimicrobial Peptide Polymyxin B1 with Both Membranes of E. coli: A Molecular Dynamics Study

    PubMed Central

    Jefferies, Damien; Sessions, Richard B.; Bond, Peter J.; Khalid, Syma

    2015-01-01

    Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development. PMID:25885324

  13. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  14. Molecular targets of (-)-epigallocatechin-3-gallate (EGCG): specificity and interaction with membrane lipid rafts.

    PubMed

    Patra, S K; Rizzi, F; Silva, A; Rugina, D O; Bettuzzi, S

    2008-12-01

    Proteomic studies on anticancer activity of Green Tea Catechins (specifically EGCG) are suggesting a large set of protein targets that may directly interact with EGCG and alter the physiology of diseased cells, including cancer. Of notice, benign cells are usually left untouched. Lipid rafts have been recently recognized as signal processing hubs and suggested to be involved in drug uptake by means of endocytosis. These findings are suggesting new insights on the molecular mechanisms of anticancer drugs action. In the membrane, EGCG is hijacked by the laminin receptor (LamR), a lipid raft protein. Similar to aplidin and edelfosin, EGCG alters membrane domains composition also preventing EGF binding to EGFR, imerization of EGFR and relocation of phosphorylated EGFR to lipid rafts. In vitro studies have recently shown that EGCG also binds both DNA and RNA in GpC-rich regions. This event may importantly affect genes function. Moreover, EGCG was shown to inhibit telomerase, topoisomerase II and DNA methyltransferase 1 (DNMT1), thus ultimately affecting chromatin maintenance and remodeling. But another important alternative pathway besides interaction with specific proteins may play an important role in EGCG action: direct targeting of bioactive membrane platforms, lipid rafts. Structural alteration of the platforms deeply impact (and often inactivates) important pathways involving MAP kinases. The key issue is that, important and specific differences in lipid rafts composition have been found in transformed versus benign cells and apoptotic versus non-apoptotic cells. We suggest here that the anticancer activity of Green Tea Catechins against different kind of cancers may find an explanation in direct targeting of lipid rafts by EGCG. PMID:19261982

  15. Interaction of antitumor alpha-lactalbumin-oleic acid complexes with artificial and natural membranes.

    PubMed

    Zherelova, Olga M; Kataev, Anatoly A; Grishchenko, Valery M; Knyazeva, Ekaterina L; Permyakov, Sergei E; Permyakov, Eugene A

    2009-06-01

    The specific complexes of human alpha-lactalbumin (alpha-LA) with oleic acid (OA), HAMLET and LA-OA-17 (OA-complexes), possess cytotoxic activity against tumor cells but the mechanism of their cell penetration remains unclear. To explore the molecular mechanisms underlying interaction of the OA-complexes with the cell membrane, their interactions with small unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles and electroexcitable plasma membrane of internodal native and perfused cells of the green alga Chara corallina have been studied. The fractionation (Sephadex G-200) of mixtures of the OA-complexes with the vesicles shows that OA-binding increases the affinity of alpha-LA to DPPC vesicles. Calcium association decreases protein affinity to the vesicles; the effect being less pronounced for LA-OA-17. The voltage clamp technique studies show that LA-OA-17, HAMLET, and their constituents produce different modifying effects on the plasmalemmal ionic channels of the Chara corallina cells. The irreversible binding of OA-complexes to the plasmalemma is accompanied by changes in the activation-inactivation kinetics of developing integral transmembrane currents, suppression of the Ca(2+) current and Ca(2+)-activated Cl(-) current, and by increase in the nonspecific K(+) leakage currents. The latter reflects development of nonselective permeability of the plasma membrane. The HAMLET-induced effects on the plasmalemmal currents are less pronounced and potentiated by LA-OA-17. The control experiments with OA and intact alpha-LA show their qualitatively different and much less pronounced effects on the transmembrane ionic currents. Thus, the modification of alpha-LA by OA results in an increase in the protein association with the model lipid bilayer and in drastic irreversible changes in permeability of several types of the plasmalemmal ionic channels.

  16. The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

    PubMed

    Palusińska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Reszczyńska, Emilia; Luchowski, Rafał; Kania, Magdalena; Gisch, Nicolas; Waldow, Franziska; Mak, Paweł; Danikiewicz, Witold; Gruszecki, Wiesław I; Cytryńska, Małgorzata

    2016-07-01

    Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III. PMID:27094351

  17. Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups*

    PubMed Central

    Yuan, Quan; Kaylor, Joanna J.; Miu, Anh; Bassilian, Sara; Whitelegge, Julian P.; Travis, Gabriel H.

    2010-01-01

    Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant. PMID:19892706

  18. The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

    PubMed

    Palusińska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Reszczyńska, Emilia; Luchowski, Rafał; Kania, Magdalena; Gisch, Nicolas; Waldow, Franziska; Mak, Paweł; Danikiewicz, Witold; Gruszecki, Wiesław I; Cytryńska, Małgorzata

    2016-07-01

    Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.

  19. Interaction of antitumor alpha-lactalbumin-oleic acid complexes with artificial and natural membranes.

    PubMed

    Zherelova, Olga M; Kataev, Anatoly A; Grishchenko, Valery M; Knyazeva, Ekaterina L; Permyakov, Sergei E; Permyakov, Eugene A

    2009-06-01

    The specific complexes of human alpha-lactalbumin (alpha-LA) with oleic acid (OA), HAMLET and LA-OA-17 (OA-complexes), possess cytotoxic activity against tumor cells but the mechanism of their cell penetration remains unclear. To explore the molecular mechanisms underlying interaction of the OA-complexes with the cell membrane, their interactions with small unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles and electroexcitable plasma membrane of internodal native and perfused cells of the green alga Chara corallina have been studied. The fractionation (Sephadex G-200) of mixtures of the OA-complexes with the vesicles shows that OA-binding increases the affinity of alpha-LA to DPPC vesicles. Calcium association decreases protein affinity to the vesicles; the effect being less pronounced for LA-OA-17. The voltage clamp technique studies show that LA-OA-17, HAMLET, and their constituents produce different modifying effects on the plasmalemmal ionic channels of the Chara corallina cells. The irreversible binding of OA-complexes to the plasmalemma is accompanied by changes in the activation-inactivation kinetics of developing integral transmembrane currents, suppression of the Ca(2+) current and Ca(2+)-activated Cl(-) current, and by increase in the nonspecific K(+) leakage currents. The latter reflects development of nonselective permeability of the plasma membrane. The HAMLET-induced effects on the plasmalemmal currents are less pronounced and potentiated by LA-OA-17. The control experiments with OA and intact alpha-LA show their qualitatively different and much less pronounced effects on the transmembrane ionic currents. Thus, the modification of alpha-LA by OA results in an increase in the protein association with the model lipid bilayer and in drastic irreversible changes in permeability of several types of the plasmalemmal ionic channels. PMID:19588235

  20. Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes.

    PubMed

    Jelokhani-Niaraki, Masoud; Hodges, Robert S; Meissner, Joseph E; Hassenstein, Una E; Wheaton, Laura

    2008-10-01

    To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic beta-sheet-beta-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar identical with Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic-exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K(b)). The K(b1) and K(b2) values for endothermic two-step binding processes corresponded to high and low binding affinities (K(b1) >or= 100 K(b2)). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.

  1. Membrane-Sugar Interactions Probed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

    PubMed

    Konov, Konstantin B; Leonov, Dmitry V; Isaev, Nikolay P; Fedotov, Kirill Yu; Voronkova, Violeta K; Dzuba, Sergei A

    2015-08-13

    Sugars can stabilize biological systems under extreme desiccation and freezing conditions. Hypothetical molecular mechanisms suggest that the stabilization effect may be determined either by specific interactions of sugars with biological molecules or by the influence of sugars on the solvating shell of the biomolecule. To explore membrane-sugar interactions, we applied electron spin echo envelope modulation (ESEEM) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), to phospholipid bilayers with spin-labeled lipids added and solvated by aqueous deuterated sucrose and trehalose solutions. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The spin-labeled lipids were 1,2-dipalmitoyl-sn-glycero-3-phospho(TEMPO)choline (T-PCSL), with spin-label TEMPO at the lipid polar headgroup. The deuterium ESEEM amplitude was calibrated using known concentrations of glassy deuterated sugar solvents. The data obtained indicated that the sugar concentration near the membrane surface obeyed a simple Langmuir model of monolayer adsorption, which assumes direct sugar-molecule bonding to the bilayer surface. PMID:26214261

  2. [Interaction of surface-active base with fraction of membrane-bound Williams's protons].

    PubMed

    Iaguzhinskiĭ, L S; Motovilov, K A; Volkov, E M; Eremeev, S A

    2013-01-01

    In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

  3. Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells.

    PubMed

    Chauveau, Anne; Aucher, Anne; Eissmann, Philipp; Vivier, Eric; Davis, Daniel M

    2010-03-23

    Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells.

  4. Structural and thermodynamic characterization of doxycycline/β-cyclodextrin supramolecular complex and its bacterial membrane interactions.

    PubMed

    Suárez, Diego F; Consuegra, Jessika; Trajano, Vivianne C; Gontijo, Sávio M L; Guimarães, Pedro P G; Cortés, Maria E; Denadai, Ângelo L; Sinisterra, Rubén D

    2014-06-01

    Doxycycline is a semi-synthetic antibiotic commonly used for the treatment of many aerobic and anaerobic bacteria. It inhibits the activity of matrix metalloproteinases (MMPs) and affects cell proliferation. In this study, the structural and thermodynamic parameters of free DOX and a DOX/βCD complex were investigated, as well as their interactions and effects on Staphylococcus aureus cells and cellular cytotoxicity. Complexation of DOX and βCD was confirmed to be an enthalpy- and entropy-driven process, and a low equilibrium constant was obtained. Treatment of S. aureus with higher concentrations of DOX or DOX/βCD resulted in an exponential decrease in S. aureus cell size, as well as a gradual neutralization of zeta potential. These thermodynamic profiles suggest that ion-pairing and hydrogen bonding interactions occur between DOX and the membrane of S. aureus. In addition, the adhesion of βCD to the cell membrane via hydrogen bonding is hypothesized to mediate a synergistic effect which accounts for the higher activity of DOX/βCD against S. aureus compared to pure DOX. Lower cytotoxicity and induction of osteoblast proliferation was also associated with DOX/βCD compared with free DOX. These promising findings demonstrate the potential for DOX/βCD to mediate antimicrobial activity at lower concentrations, and provides a strategy for the development of other antimicrobial formulations.

  5. Demonstration of the interactions between aromatic compound-loaded lipid nanocapsules and Acinetobacter baumannii bacterial membrane.

    PubMed

    Montagu, A; Joly-Guillou, M-L; Guillet, C; Bejaud, J; Rossines, E; Saulnier, P

    2016-06-15

    Acinetobacter baumannii is an important nosocomial pathogen that is resistant to many commonly-used antibiotics. One strategy for treatment is the use of aromatic compounds (carvacrol, cinnamaldehyde) against A. baumannii. The aim of this study was to determine the interactions between bacteria and lipid nanocapsules (LNCs) over time based on the fluorescence of 3,3'-Dioctadecyloxacarbocyanine Perchlorate-LNCs (DiO-LNCs) and the properties of trypan blue to analyse the physicochemical mechanisms occurring at the level of the biological membrane. The results demonstrated the capacity of carvacrol-loaded LNCs to interact with and penetrate the bacterial membrane in comparison with cinnamaldehyde-loaded LNCs and unloaded LNCs. Modifications of carvacrol after substitution of hydroxyl functional groups by fatty acids demonstrated the crucial role of hydroxyl functions in antibacterial activity. Finally, after contact with the efflux pump inhibitor, carbonylcyanide-3-chlorophenyl hydrazine (CCCP), the results indicated the total synergistic antibacterial effect with Car-LNCs, showing that CCCP is associated with the action mechanism of carvacrol, especially at the level of the efflux pump mechanism. PMID:27039148

  6. X-ray scattering study of the interactions between magnetic nanoparticles and living cell membranes

    SciTech Connect

    Koh, Isaac; Cipriano, Bani H.; Ehrman, Sheryl H.; Williams, Darryl N.; Pulliam Holoman, Tracey R.; Martinez-Miranda, L. J.

    2005-04-15

    Magnetic nanoparticles (MNPs) have found increased applicability in drug delivery, cancer treatment, and immunoassays. There is a need for an improved understanding of how MNPs interact with living cell membranes in applied magnetic fields to use them effectively. The interactions between Escherichia coli (E. coli) and SiO{sub 2}/{gamma}-Fe{sub 2}O{sub 3} composite particles in magnetic fields were studied using x-ray scattering. Magnetic field strengths up to 423 mT were applied to the samples to see the effects of the magnetic fields on the E. coli membranes in the presence of the magnetic particles in the cell cultures. X-ray scattering results from continuous cultures of E. coli showed two peaks, a sharp peak at q=0.528 A{sup -1} (1.189 nm) up to 362 mT of magnetic field strength and a diffuse one at q=0.612 A{sup -1} (1.027 nm). The sharp peak was shifted to the smaller side of q when magnetic particles were added and the magnitude of the applied magnetic field strength was increased from 227 to 298 mT, to 362 mT, whereas the diffuse peak did not changed. A critical magnetic field strength where the sharp peak disappears was found at 362 mT.

  7. Dynamic assessment of Amyloid oligomers - cell membrane interaction by advanced impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Gheorghiu, M.; David, S.; Polonschii, C.; Bratu, D.; Gheorghiu, E.

    2013-04-01

    The amyloid β (Aβ) peptides are believed to be pivotal in Alzheimer's disease (AD) pathogenesis and onset of vascular dysfunction. Recent studies indicate that Aβ1-42 treatment influences the expression of tight junction protein complexes, stress fibre formation, disruption and aggregation of actin filaments and cellular gap formation. Aiming for functional characterization of model cells upon Aβ1-42 treatment, we deployed an advanced Electric Cell-substrate Impedance Sensing for monitoring cell evolution. A precision Impedance Analyzer with a multiplexing module developed in house was used for recording individual electrode sets in the 40 Hz - 100 KHz frequency range. In a step forward from the classical ECIS assays, we report on a novel data analysis algorithm that enables access to cellular and paracellular electrical parameters and cell surface interaction with fully developed cell monolayers. The evolution of the impedance at selected frequencies provides evidence for a dual effect of Aβ42 exposure, at both paracellular permeability and cell adherence level, with intricate dynamics that open up new perspectives on Aβ1-42 oligomers - cell membrane interaction. Validation of electrical impedance assays of the amyloid fibrils effect on cell membrane structure is achieved by both AFM analysis and Surface Plasmon Resonance studies. The capabilities of this noninvasive, real time platform for cell analysis in a wider applicative context are outlined.

  8. Hydrophobic ion interactions with membranes. Thermodynamic analysis of tetraphenylphosphonium binding to vesicles.

    PubMed Central

    Flewelling, R F; Hubbell, W L

    1986-01-01

    The thermodynamic properties for the interaction of the hydrophobic ion tetraphenylphosphonium (TPP+) with egg phosphatidylcholine vesicles were studied in detail by equilibrium dialysis and spin label techniques. A partition coefficient of beta = 4.2 + 0.4 x 10(-6) cm (K congruent to 100) was determined. Electrostatic saturation sets in at approximately 600 microM (about one absorbed TPP+ molecule per 100 lipids), and is not screened by salt. The temperature dependence of binding was determined, which reveals that the binding is entropy-driven with a positive (repulsive) enthalpy of binding, a result to be compared with hydrophobic anions in which the binding enthalpy is negative. The membrane dipole potential may be responsible for this binding difference. Activity coefficients are determined and shown to be significantly different from those of most common salts, an important result that should be considered in all hydrophobic ion studies. Comparison of the TPP+ results with those of its anionic structural analogue, tetraphenylboron (TPB-), permits a general analysis of hydrophobic ion interactions with membranes. A theoretical model consistent with the entire set of data is developed in an accompanying article. PMID:3006814

  9. Interaction of plasma fibronectin (pFN) with membranous constituents of peritoneal exudate cells and pulmonary macrophages

    SciTech Connect

    Rovin, B.; Molnar, J.; Chevalier, D.; Ng, P.

    1984-11-01

    The prominent role of plasma fibronectin (pFN) in the host defense system as an opsonin for gelatin (collagen)-coated colloids has been established. In the present study the authors investigated the interaction of pFN and membrane isolates from cells devoid of collagen, as well as several tissues. In a liver slice assay system it was shown that subcellular membrane fractions from lung macrophages, peritoneal exudate cells, spleen, testis, and liver were able to competitively inhibit the pFN-mediated uptake of /sup 125/I-gelatin coated latex beads (gLtx) at low concentrations. Endocytosis of /sup 125/I-labeled membrane isolates by macrophage monolayers was also promoted by addition of pFN. In an attempt to characterize the membrane component(s) interacting with pFN, it was found that mild extraction procedure with 1 M KBr could release a significant amount of this inhibitory activity. Further studies demonstrated that the agent(s) responsible for inhibition of gLtx uptake was heat sensitive, not altered by trypsin treatment, and did not contain actin, a protein known to interact with pFN. This work indicates that pFN interacts specifically with an as yet unknown membrane component(s) and that such interaction will promote clearance of cellular debris by macrophages. This suggests that pFN may be an important opsonin for the reticuloendothelial system in clearance of collagenous and noncollagenous cellular debris once they are exposed to interact with it.

  10. Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

    PubMed Central

    Wong, Siew Heng; Zhang, Tao; Xu, Yue; Subramaniam, V. Nathan; Griffiths, Gareth; Hong, Wanjin

    1998-01-01

    Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of th