Lipid nanotechnologies for structural studies of membrane-associated proteins.

Science.gov (United States)

Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

2014-11-01

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

Monitoring orientation and dynamics of membrane-bound melittin utilizing dansyl fluorescence.

Science.gov (United States)

Haldar, Sourav; Raghuraman, H; Chattopadhyay, Amitabha

2008-11-06

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. In spite of a number of studies, there is no consensus regarding the orientation of melittin in membranes. In this study, we used a melittin analogue that is covalently labeled at its amino terminal (Gly-1) with the environment-sensitive 1-dimethylamino-5-sulfonylnaphthalene (dansyl) group to obtain information regarding the orientation and dynamics of the amino terminal region of membrane-bound melittin. Our results show that the dansyl group in Dns-melittin exhibits red edge excitation shift in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine, implying its localization in a motionally restricted region of the membrane. This is further supported by wavelength-dependent anisotropy and lifetime changes and time-resolved emission spectra characterized by dynamic Stokes shift, which indicates relatively slow solvent relaxation in the excited state. Membrane penetration depth analysis using the parallax method shows that the dansyl group is localized at a depth of approximately 18 A from the center of the bilayer in membrane-bound Dns-melittin. Further analysis of dansyl and tryptophan depths in Dns-melittin shows that the tilt angle between the helix axis of membrane-bound melittin and the bilayer normal is approximately 70 degrees. Our results therefore suggest that melittin adopts a pseudoparallel orientation in DOPC membranes at low concentration.

Crystal structure of Na+, K(+)-ATPase in the Na(+)-bound state.

Science.gov (United States)

Nyblom, Maria; Poulsen, Hanne; Gourdon, Pontus; Reinhard, Linda; Andersson, Magnus; Lindahl, Erik; Fedosova, Natalya; Nissen, Poul

2013-10-04

The Na(+), K(+)-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na(+) and K(+) across the plasma membrane--a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K(+)-bound or ouabain-blocked forms. We present the crystal structure of a Na(+)-bound Na(+), K(+)-ATPase as determined at 4.3 Å resolution. Compared with the K(+)-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na(+) sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na(+) from IIIb through IIIa, followed by exchange of Na(+) for K(+) at sites I and II, is suggested.

Tumor promoter induced membrane-bound protein kinase C - its influence on hematogenous metastasis

International Nuclear Information System (INIS)

Gopalakrishna, R.; Barsky, S.H.

1987-01-01

A correlation between the amount of membrane-bound detergent-extractable protein kinase C activity in various B16 melanoma sublines (F10, F1, BL6) and their lung metastasizing abilities following intravenous injection was found. The F10 subline which exhibits higher metastasizing ability was found to have higher membrane-bound protein kinase C compared to the lower metastasizing subline, F1. Treatment of F1 cells with 100 nM 12-0 tetradecanoylphorbol-13-acetate (TPA) for 1h resulted in 90% decrease in protein kinase C activity in the cytosol with a concommitent increase in membrane-bound activity. These TPA-treated cells when injected intravenously in C57BL/6 mice produced 6-fold increase in pulmonary metastases compared to untreated F1 cells. However, biologically inactive analogues 4 α-phorbol 12,13-didecanoate and phorbol 13-acetate had no effect on either membrane-bound protein kinase C activity or pulmonary metastases. Treating F1 cells with the second-stage tumor promoter, mezerin, resulted in increase in both membrane association of protein kinase C and also lung metastases. Thus, these results strongly suggests that membrane associated protein kinase C activity influences hematogenous metastasis of these melanoma cells

Heterologous expression and purification of membrane-bound pyrophosphatases

DEFF Research Database (Denmark)

Kellosalo, J.; Kajander, T.; Palmgren, Michael Broberg

2011-01-01

Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low l...

TUNABLE TENSOR VOTING FOR REGULARIZING PUNCTATE PATTERNS OF MEMBRANE-BOUND PROTEIN SIGNALS

OpenAIRE

Loss, Leandro

2009-01-01

Membrane-bound protein, expressed in the basal-lateral region, is heterogeneous and an important endpoint for understanding biological processes. At the optical resolution, membrane-bound protein can be visualized as being diffused (e.g., E-cadherin), punctate (e.g., connexin), or simultaneously diffused and punctate as a result of sample preparation or conditioning. Furthermore, there is a significant amount of heterogeneity as a result of technical and biological variations. This paper aims...

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

Science.gov (United States)

Schröter, W; Neuvians, M

1970-12-01

Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

International Nuclear Information System (INIS)

Belkin, V.M.; Volodarskaya, S.M.

1986-01-01

The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes

Recognition of GPCRs by peptide ligands and membrane compartments theory: structural studies of endogenous peptide hormones in membrane environment.

Science.gov (United States)

Sankararamakrishnan, Ramasubbu

2006-04-01

One of the largest family of cell surface proteins, G-protein coupled receptors (GPCRs) regulate virtually all known physiological processes in mammals. With seven transmembrane segments, they respond to diverse range of extracellular stimuli and represent a major class of drug targets. Peptidergic GPCRs use endogenous peptides as ligands. To understand the mechanism of GPCR activation and rational drug design, knowledge of three-dimensional structure of receptor-ligand complex is important. The endogenous peptide hormones are often short, flexible and completely disordered in aqueous solution. According to "Membrane Compartments Theory", the flexible peptide binds to the membrane in the first step before it recognizes its receptor and the membrane-induced conformation is postulated to bind to the receptor in the second step. Structures of several peptide hormones have been determined in membrane-mimetic medium. In these studies, micelles, reverse micelles and bicelles have been used to mimic the cell membrane environment. Recently, conformations of two peptide hormones have also been studied in receptor-bound form. Membrane environment induces stable secondary structures in flexible peptide ligands and membrane-induced peptide structures have been correlated with their bioactivity. Results of site-directed mutagenesis, spectroscopy and other experimental studies along with the conformations determined in membrane medium have been used to interpret the role of individual residues in the peptide ligand. Structural differences of membrane-bound peptides that belong to the same family but differ in selectivity are likely to explain the mechanism of receptor selectivity and specificity of the ligands. Knowledge of peptide 3D structures in membrane environment has potential applications in rational drug design.

Impact of the antimicrobial peptide Novicidin on membrane structure and integrity

DEFF Research Database (Denmark)

Nielsen, Søren B; Otzen, Daniel Erik

2010-01-01

We have studied the impact of an 18-residue cationic antimicrobial peptide Novicidin (Nc) on the structure and integrity of partially anionic lipid membranes using oriented circular dichroism (OCD), quartz crystal microbalance with dissipation (QCM-D), dual polarization interferometry (DPI......), calcein dye leakage and fluorescence spectroscopy. OCD consistently showed that Nc is bound in an alpha-helical, surface bound state over a range of peptide to lipid (P/L) ratios up to approximately 1:15. Realignment of Nc at higher P/L ratios correlates to loss of membrane integrity as shown by Laurdan...... concentration, probably through formation of transient pores or transient disruption of the membrane integrity, followed by more extensive membrane disintegration at higher P/L ratios....

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

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Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

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

Membrane-bound transcription factors: regulated release by RIP or RUP.

Science.gov (United States)

Hoppe, T; Rape, M; Jentsch, S

2001-06-01

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Carlo Barnaba

2017-05-01

Full Text Available Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b5—are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

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

Science.gov (United States)

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

2010-02-23

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

Response of membrane-bound ATPase of Micrococcus luteus to heat and ultraviolet light

International Nuclear Information System (INIS)

Volotovskij, J.; Risi, S.; Dose, K.

1976-01-01

It is shown that the properties of ATPase (EC 3.6.1.3) of Micrococcus luteus depend only to some extent on the state of the membrane to which it is attached. Its interaction with the membrane appears to be largely controlled by polar forces. It is shown, however, that the UV-sensitivity of the membrane-bound ATPase is also significantly influenced by the state of membrane lipids. (orig.) [de

Response of membrane-bound ATPase of Micrococcus luteus to heat and ultraviolet light

Energy Technology Data Exchange (ETDEWEB)

Volotovskii, J; Risi, S; Dose, K [Mainz Univ. (F.R. Germany). Inst. fuer Biochemie

1976-03-01

It is shown that the properties of ATPase (EC 3.6.1.3) of Micrococcus luteus depend only to some extent on the state of the membrane to which it is attached. Its interaction with the membrane appears to be largely controlled by polar forces. It is shown, however, that the UV-sensitivity of the membrane-bound ATPase is also significantly influenced by the state of membrane lipids.

Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

NARCIS (Netherlands)

Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

2002-01-01

The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

Study on the changes in the levels of membrane-bound ATPases ...

African Journals Online (AJOL)

An attempt has been made to determine the deleterious effects of λ cyhalothrin- induced in fresh water tilapia (Oreochromis mossambicus) with respect to changes in the activities of membrane-bound ATPases (Na+/K+, Mg+ and Ca2+ ATPase) and mineral status ...

A new look at lipid-membrane structure in relation to drug research

DEFF Research Database (Denmark)

Mouritsen, Ole G.; Jørgensen, Kent

1998-01-01

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

Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

International Nuclear Information System (INIS)

Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

2008-01-01

Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

Directory of Open Access Journals (Sweden)

Manetta J

2014-08-01

Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach.

Science.gov (United States)

Murata, Michio; Sugiyama, Shigeru; Matsuoka, Shigeru; Matsumori, Nobuaki

2015-08-01

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology. © 2015 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

How membrane lipids control the 3D structure and function of receptors

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Jacques Fantini

2018-02-01

Full Text Available The cohabitation of lipids and proteins in the plasma membrane of mammalian cells is controlled by specific biochemical and biophysical rules. Lipids may be either constitutively tightly bound to cell-surface receptors (non-annular lipids or less tightly attached to the external surface of the protein (annular lipids. The latter are exchangeable with surrounding bulk membrane lipids on a faster time scale than that of non-annular lipids. Not only do non-annular lipids bind to membrane proteins through stereoselective mechanisms, they can also help membrane receptors acquire (or maintain a functional 3D structure. Cholesterol is the prototype of membrane lipids that finely controls the 3D structure and function of receptors. However, several other lipids such as sphingolipids may also modulate the function of membrane proteins though conformational adjustments. All these concepts are discussed in this review in the light of representative examples taken from the literature.

Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

DEFF Research Database (Denmark)

Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas

2017-01-01

Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosyla......Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes...

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

Science.gov (United States)

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-01-01

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K+·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K+, in marked contrast to previous models. Due to antagonism between ouabain and K+, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity. PMID:19666591

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain.

Science.gov (United States)

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-08-18

The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

DEFF Research Database (Denmark)

Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner

2011-01-01

and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane...... of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²5I-rFVIII) and annexin V...... or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could...

Membrane Structure Studies by Means of Small-Angle Neutron Scattering (SANS)

International Nuclear Information System (INIS)

Knott, R. B.

2008-01-01

The basic model for membrane structure--a lipid bilayer with imbedded proteins--was formulated 35 years ago, however the detailed structure is still under active investigation using a variety of physical, chemical and computational techniques. Every biologically active cell is encapsulated by a plasma membrane with most cells also equipped with an extensive intracellular membrane system. The plasma membrane is an important boundary between the cytoplasm of the cell and the external environment, and selectively isolates the cell from that environment. Passive diffusion and/or active transport mechanisms are provided for water, ions, substrates etc. which are vital for cell metabolism and viability. Membranes also facilitate excretion of substances either as useful cellular products or as waste. Despite their complexity and diverse function, plasma membranes from quite different cells have surprisingly similar compositions. A typical membrane structure consists of a phospholipid bilayer with a number of proteins scattered throughout, along with carbohydrates (glycoproteins), glycolipids and sterols. The plasma membranes of most eukaryotic cells contain approximately equal weights of lipid and protein, which corresponds to about 100 lipid molecules per protein molecule. Clearly, lipids are a major constituent and the study of their structure and function in isolation provides valuable insight into the more complex intact multicomponent membrane. The membrane bound protein is the other major constituent and is a very active area of research for a number of reasons including the fact that over 60% of modern drugs act on their receptor sites. The interaction between the protein and the supporting lipid bilayer is clearly of major importance. Neutron scattering is a powerful technique for exploring the structure of membranes, either as reconstituted membranes formed from well characterised lipids, or as intact membranes isolated from selected biological systems. A brief

Characterization and expression patterns of a membrane-bound trehalase from Spodoptera exigua

Directory of Open Access Journals (Sweden)

Xu Weihua

2008-05-01

Full Text Available Abstract Background The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1 and membrane-bound trehalase (Tre-2, very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2. Results To gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from Spodoptera exigua (SeTre-2 cloned from the fat body. The deduced amino acid sequence of SeTre-2 contains 645 residues and has a predicted molecular weight of ~74 kDa and pI of 6.01. Alignment of SeTre-2 with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of Tre-2. Comparison of the genomic DNA and cDNA sequences demonstrated that SeTre-2 comprises 13 exons and 12 introns. Southern blot analysis revealed that S. exigua has two trehalase genes and that SeTre-2 is a single-copy gene. Northern blot analyses showed that the SeTre-2 transcript is expressed not only in the midgut, as previously reported for Bombyx mori, but also in the fat body and Malpighian tubules, although expression patterns differed between the midgut and fat body. SeTre-2 transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas SeTre-2 mRNA was detected in the fat body of fifth instar larvae and pupae. Conclusion These findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. The results suggest that the SeTre-2 gene may have different functions in the midgut and fat body.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Directory of Open Access Journals (Sweden)

Anna Lundin

2014-05-01

Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Paramagnetic relaxation enhancements in NMR peptide-membrane interaction studies

International Nuclear Information System (INIS)

Kosol, S.

2011-01-01

Small membrane-bound proteins or peptides are involved in numerous essential biological processes, like cellular recognition, signaling, channel formation, and cytolysis. The secondary structure, orientation, mode of interaction and dynamics of these peptides can be as varied as their functions. Their localization in the membrane, the immersion depth, and their binding mode are factors critical to the function of these peptides. The atomic 3D solution structure of peptides bound to micelles can be determined by NMR spectroscopy. However, by employing paramagnetic relaxation enhancements (PREs) information on the complete topology of peptide bound to a micelle can be obtained. The antimicrobial peptide maximin H6, fst, a bacterial toxin, and the human peptide hormone ghrelin served as membrane-bound model peptides of similar sizes but strongly differing amino acid sequences. Their structures and binding behavior were determined and compared.The measured PREs provided suitable data for determining and distinguishing the different topologies of the investigated peptides bound to micelles. Maximin H6 and fst fold into α-helices upon insertion into a membrane, whereas the unstructured ghrelin is freely mobile in solution and interacts only via a covalently bound octanoyl group with the lipids. Maximin H6 is oriented parallel to the membrane surface, enabling the peptide to aggregate at the membrane water interface. Fst binds in transmembrane orientation with a protruding intrinsically disordered region near the C-terminus. Aside from determining the orientation of the bound peptides from the PREs, the moieties critical for membrane binding could be mapped in ghrelin. If suitable relaxation-edited spectra are acquired, the complete orientation and immersion depth of a peptide bound to a micelle can readily be obtained. (author) [de

Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans

International Nuclear Information System (INIS)

Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R.; Chiang, Janet; Gunsalus, Robert P.; Arbing, Mark A.; Perry, L. Jeanne

2010-01-01

Crystal structures of ModA from M. acetivorans in the apo and ligand-bound conformations confirm domain rotation upon ligand binding. The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 Å resolution and in the molybdate-bound conformation at 2.25 and 2.45 Å resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed α/β domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the ‘Venus flytrap’ model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins

Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats.

Science.gov (United States)

Al-Numair, Khalid S; Veeramani, Chinnadurai; Alsaif, Mohammed A; Chandramohan, Govindasamy

2015-01-01

Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities. The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40 mg/kg body weight (BW)). Kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) was administered orally once daily for 45 d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined. In our study, diabetic rats had significantly (p kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) for a period of 45 d resulted in significant (p kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol's action mechanism.

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study.

Directory of Open Access Journals (Sweden)

Huai-Chun Chen

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

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

Directory of Open Access Journals (Sweden)

Yihao Zhang

2017-02-01

Full Text Available Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav, which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

Science.gov (United States)

Zhang, Yihao; Abiraman, Krithika; Li, He; Pierce, David M; Tzingounis, Anastasios V; Lykotrafitis, George

2017-02-01

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

International Nuclear Information System (INIS)

Wylie, Benjamin J.; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

2015-01-01

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces

Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone.

Science.gov (United States)

Sampedro, Javier; Valdivia, Elene R; Fraga, Patricia; Iglesias, Natalia; Revilla, Gloria; Zarra, Ignacio

2017-02-01

In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked β-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) β-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for β-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound β-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer. © 2017 American Society of Plant Biologists. All Rights Reserved.

Bounds on the Effect of Progressive Structural Degradation

DEFF Research Database (Denmark)

Achtziger, W.; Bendsøe, Martin P; Taylor, John E.

1998-01-01

Problem formulations are presented for the evaluation of upper and lower bounds on the effect of progressive structural degradation. For the purposes of this study, degradation effect is measured by an increase in global structural compliance (flexibility). Thus the slated bounds are given simply...

Bounds on the Effect of Progressive Structural Degradation

DEFF Research Database (Denmark)

Achtziger, Wolfgang; Bendsøe, Martin P; Taylor, John E.

1997-01-01

Problem formulations are presented for the evaluation of upper and lower bounds on the effect of progressive structural degradation. For the purposes of this study, degradation effect is measured by an increase in global structural compliance (flexibility). Thus the stated bounds are given simply...

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

OpenAIRE

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-01-01

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analog...

Membrane-associated insulin-like growth factor (IGF binding structures in placental cells

Directory of Open Access Journals (Sweden)

ROMANA MASNIKOSA

2003-11-01

Full Text Available The biological activities of IGF-I and –II are mediated mainly by the type 1 IGF receptor (IGF 1R and controlled by their interaction with soluble proteins, the IGF binding proteins (IGFBPs. Although there is a growing body of evidence that some IGFBPs may be cell surface-bound, published data concerning cell association of IGFBP-1 are scarce and none of them concern placental cells. The cell membranes used in this study were isolated from term human placentae. Detergent-solubilized membranes were shown to contain two types of IGF binding structures that were separated by gel filtration on a Sephadex G-100 column. Proteins in the first peak were eluted at V0 (Mr > 100 kD and they bound IGF-I with greater specificity and affinity than IGF-II and insulin. Most likely, they represented the IGF 1R. Small proteins (Mr ~ 45 kD were eluted with the membrane proteins in the second maximum. They were able to bind IGF-I and IGF-II, but not insulin. The identity of these proteins was shown to be IGFBP-1 on the basis of their reaction with specific anti-IGFBP-1 antibodies. To the best of our knowledge, the existence of IGFBP-1 associated with human placental cell membranes has not been reported in the literature before. Colocalisation of IGFBP-1 with IGF 1R in cell membranes could provide efficient modulation of IGF 1R receptor-ligand interactions.

Models and Techniques for Proving Data Structure Lower Bounds

DEFF Research Database (Denmark)

Larsen, Kasper Green

In this dissertation, we present a number of new techniques and tools for proving lower bounds on the operational time of data structures. These techniques provide new lines of attack for proving lower bounds in both the cell probe model, the group model, the pointer machine model and the I...... bound of tutq = (lgd􀀀1 n). For ball range searching, we get a lower bound of tutq = (n1􀀀1=d). The highest previous lower bound proved in the group model does not exceed ((lg n= lg lg n)2) on the maximum of tu and tq. Finally, we present a new technique for proving lower bounds....../O-model. In all cases, we push the frontiers further by proving lower bounds higher than what could possibly be proved using previously known techniques. For the cell probe model, our results have the following consequences: The rst (lg n) query time lower bound for linear space static data structures...

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

Energy Technology Data Exchange (ETDEWEB)

Cady, Sarah D.; Hong Mei [Iowa State University, Department of Chemistry (United States)], E-mail: mhong@iastate.edu

2009-09-15

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the {sup 1}H rotating-frame spin-lattice relaxation (T{sub 1{rho}}) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by {sup 2}H, {sup 15}N and {sup 13}C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent {sup 1}H T{sub 1{rho}} relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 {+-} 0.9 {mu}s for the apo peptide to 0.89 {+-} 0.41 {mu}s for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of ln (T{sub 1{rho}}{sup -1}) indicates that the activation energy of motion increased from 14.0 {+-} 4.0 kJ/mol for the apo peptide to 23.3 {+-} 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.

Structure and physical properties of bio membranes and model membranes

International Nuclear Information System (INIS)

Tibor Hianik

2006-01-01

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

Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

Science.gov (United States)

Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

2017-11-07

The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a membrane-bound, glycol-stimulated phospholipase A2 located in the secretory granules of the adrenal medulla

International Nuclear Information System (INIS)

Hildebrandt, E.; Albanesi, J.P.

1991-01-01

Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca 2+ -stimulated phospholipase A 2 (PLA 2 ) activity when assayed at pH 9.0 with phosphatidylcholine containing an [ 14 C]-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA 2 activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycole, or poly(ethylene glycol). This glycol-stimulated PLA 2 activity codistributed with membrane-bound dopamine β-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA 2 of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different 14 C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethhanolamine, and distearoylphosphatidylcholine was not hydrolyzed

Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

International Nuclear Information System (INIS)

Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

1987-01-01

In this work, we examined occlusion of 22 Na+ and 86 Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of 22 Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of 22 Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by 32 P incorporation from [gamma- 32 P]CrATP. Maximum capacity for phosphorylation from [gamma- 32 P]CrATP was 6 nmol/mg of protein and equal to capacities for binding of [48V]vanadate and [ 3 H]ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport

Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

Science.gov (United States)

Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R; Chiang, Janet; Gunsalus, Robert P; Arbing, Mark A; Perry, L Jeanne

2010-03-01

The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

Science.gov (United States)

Hong, Mei; Su, Yongchao

2011-01-01

Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

Science.gov (United States)

Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Albumin solder covalently bound to a polymer membrane: New approach to improve binding strength in laser tissue soldering in-vitro.

Science.gov (United States)

Hiebl, B; Ascher, L; Luetzow, K; Kratz, K; Gruber, C; Mrowietz, C; Nehring, M E; Lendlein, A; Franke, R-P; Jung, F

2018-01-01

Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat-denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG-supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250 mW and 1500 mW was utilized. The albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2. All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450 mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002 MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1 mm around the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native

Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate.

Science.gov (United States)

Manzo, Anthony J; Goushcha, Alexander O; Barabash, Yuri M; Kharkyanen, Valery N; Scott, Gary W

2009-07-01

Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I(exp), obeys a simple exponential law with the rate constant alphaI(exp) + k(rec), in which alpha is a parameter relating the light intensity, measured in mW/cm(2), to a corresponding theoretical rate in units of reciprocal seconds, and k(rec) is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the alpha parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer-Lambert-Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

Science.gov (United States)

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

Redshift-space limits of bound structures

NARCIS (Netherlands)

Duenner, Rolando; Reisenegger, Andreas; Meza, Andres; Araya, Pablo A.; Quintana, Hernan

2007-01-01

An exponentially expanding Universe, possibly governed by a cosmological constant, forces gravitationally bound structures to become more and more isolated, eventually becoming causally disconnected from each other and forming so-called 'island universes'. This new scenario reformulates the question

[The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

Science.gov (United States)

Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

2016-01-01

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

Membrane interaction and secondary structure of de novo designed arginine-and tryptophan peptides with dual function

KAUST Repository

Rydberg, Hanna A.

2012-10-01

Cell-penetrating peptides and antimicrobial peptides are two classes of positively charged membrane active peptides with several properties in common. The challenge is to combine knowledge about the membrane interaction mechanisms and structural properties of the two classes to design peptides with membrane-specific actions, useful either as transporters of cargo or as antibacterial substances. Membrane active peptides are commonly rich in arginine and tryptophan. We have previously designed a series of arg/trp peptides and investigated how the position and number of tryptophans affect cellular uptake. Here we explore the antimicrobial properties and the interaction with lipid model membranes of these peptides, using minimal inhibitory concentrations assay (MIC), circular dichroism (CD) and linear dichroism (LD). The results show that the arg/trp peptides inhibit the growth of the two gram positive strains Staphylococcus aureus and Staphylococcus pyogenes, with some individual variations depending on the position of the tryptophans. No inhibition of the gram negative strains Proteus mirabilis or Pseudomonas aeruginosa was noticed. CD indicated that when bound to lipid vesicles one of the peptides forms an α-helical like structure, whereas the other five exhibited rather random coiled structures. LD indicated that all six peptides were somehow aligned parallel with the membrane surface. Our results do not reveal any obvious connection between membrane interaction and antimicrobial effect for the studied peptides. By contrast cell-penetrating properties can be coupled to both the secondary structure and the degree of order of the peptides. © 2012 Elsevier Inc.

A membrane-bound vertebrate globin.

Directory of Open Access Journals (Sweden)

Miriam Blank

Full Text Available The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2 binding with a variable affinity (P(50∼1.3-12.5 torr, depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

Bound-state quark and gluon contributions to structure functions in QCD

International Nuclear Information System (INIS)

Brodsky, S.J.

1991-01-01

One can distinguish two types of contributions to the quark and gluon structure functions of hadrons in quantum chromodynamics: 'intrinsic' contributions, which are due to the direct scattering on the bound-state constituents, and 'extrinsic' contributions, which are derived from particles created in the collision. In this talk, I discuss several aspects of deep inealstic structure functions in which the bound-state structure of the proton plays a crucial role: (1) the properties of the intrinsic gluon distribution associated with the proton bound-state wavefunction; (2) the separation of the quark structure function of the proton into intrinsic 'bound-valence' and extrinsic 'non-valence' components which takes into account the Pauli principle; (3) the properties and identification of intrinsic heavy quark structure functions; and (4) a theory of shadowing and anti-shadowing of nuclear structure functions, directly related to quark-nucleon interactions and the gluon saturation phenomenon. (orig.)

Bound-state quark and gluon contributions to structure functions in QCD

International Nuclear Information System (INIS)

Brodsky, S.J.

1990-08-01

One can distinguish two types of contributions to the quark and gluon structure functions of hadrons in quantum chromodynamics: ''intrinsic'' contributions, which are due to the direct scattering on the bound-state constituents, and ''extrinsic'' contributions, which are derived from particles created in the collision. In this talk, I discussed several aspects of deep inelastic structure functions in which the bound-state structure of the proton plays a crucial role: the properties of the intrinsic gluon distribution associated with the proton bound-state wavefunction; the separation of the quark structure function of the proton onto intrinsic ''bound-valence'' and extrinsic ''non-valence'' components which takes into account the Pauli principle; the properties and identification of intrinsic heavy quark structure functions; and a theory of shadowing and anti-shadowing of nuclear structure functions, directly related to quark-nucleon interactions and the gluon saturation phenomenon. 49 refs., 5 figs

Thermodynamic Upper Bound on Broadband Light Coupling with Photonic Structures

KAUST Repository

Yu, Zongfu; Raman, Aaswath; Fan, Shanhui

2012-01-01

to an upper bound dictated by the second law of thermodynamics. Such bound limits how efficient light can be coupled to any photonic structure. As one example of application, we use this upper bound to derive the limit of light absorption in broadband solar

Review of Large Spacecraft Deployable Membrane Antenna Structures

Science.gov (United States)

Liu, Zhi-Quan; Qiu, Hui; Li, Xiao; Yang, Shu-Li

2017-11-01

The demand for large antennas in future space missions has increasingly stimulated the development of deployable membrane antenna structures owing to their light weight and small stowage volume. However, there is little literature providing a comprehensive review and comparison of different membrane antenna structures. Space-borne membrane antenna structures are mainly classified as either parabolic or planar membrane antenna structures. For parabolic membrane antenna structures, there are five deploying and forming methods, including inflation, inflation-rigidization, elastic ribs driven, Shape Memory Polymer (SMP)-inflation, and electrostatic forming. The development and detailed comparison of these five methods are presented. Then, properties of membrane materials (including polyester film and polyimide film) for parabolic membrane antennas are compared. Additionally, for planar membrane antenna structures, frame shapes have changed from circular to rectangular, and different tensioning systems have emerged successively, including single Miura-Natori, double, and multi-layer tensioning systems. Recent advances in structural configurations, tensioning system design, and dynamic analysis for planar membrane antenna structures are investigated. Finally, future trends for large space membrane antenna structures are pointed out and technical problems are proposed, including design and analysis of membrane structures, materials and processes, membrane packing, surface accuracy stability, and test and verification technology. Through a review of large deployable membrane antenna structures, guidance for space membrane-antenna research and applications is provided.

Electromagnetic structure of a bound nucleon

International Nuclear Information System (INIS)

Nogami, Y.

1977-01-01

The effect of binding on the electromagnetic (e.m.) structure of a nucleon in a nucleus is examined by means of a model consisting of a single nucleon which is bound in a harmonic oscillator potential and also coupled to the pion field through the Chew-Low interaction. The 'two-pion contribution' to the e.m. structure is considered. This is the part which is probably most susceptible to the binding effect. By the binding effect it is meant the one which arises because the nucleon wave functions, in the intermediate state as well as in the initial and final states, are distorted by the potential in which the nucleon is bound. This may be compared to a similar correction to the impulse approximation for pion-nucleus scattering. Unlike the latter which is likely to be quite appreciable, the binding correction to the e.m. structure of the nucleon is found to be negligibly small. The so-called quenching effect due to the Pauli principle when there are other nucleons is also discussed [pt

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level

Science.gov (United States)

Belyavskaya, N. A.

The changes of [Ca^2+]_i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station ``Salyut 6'' /1/. These results: 1) indicate that observed Ca^2+-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca^2+ influx through membranes. In model presented, I propose that Ca^2+-activated channels in plasma membrane in response to microgravity allow the movement of Ca^2+ into the root cells, causing a rise in cytoplasmic free Ca^2+ levels. The latter, in its turn, may induce the inhibition of a Ca^2+ efflux by Ca^2+-activated ATPases and through a Ca^2+/H^+ antiport. It is possible that increased cytosolic levels of Ca^2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca^2+]_i. Plant cell can response to such a Ca^2+ rise by an enhancement of membranous Ca^2+-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca^2+-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca^2+ to plant cell.

Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

Energy Technology Data Exchange (ETDEWEB)

Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

2008-10-22

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

Structure and properties of cell membranes. Volume 3: Methodology and properties of membranes

International Nuclear Information System (INIS)

Benga, G.

1985-01-01

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

Thermodynamic Upper Bound on Broadband Light Coupling with Photonic Structures

KAUST Repository

Yu, Zongfu

2012-10-01

The coupling between free space radiation and optical media critically influences the performance of optical devices. We show that, for any given photonic structure, the sum of the external coupling rates for all its optical modes are subject to an upper bound dictated by the second law of thermodynamics. Such bound limits how efficient light can be coupled to any photonic structure. As one example of application, we use this upper bound to derive the limit of light absorption in broadband solar absorbers. © 2012 American Physical Society.

Probing membrane protein structure using water polarization transfer solid-state NMR.

Science.gov (United States)

Williams, Jonathan K; Hong, Mei

2014-10-01

Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected (1)H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins. Copyright © 2014 Elsevier Inc. All

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

International Nuclear Information System (INIS)

Moaddel, Ruin; Wainer, Irving W.

2006-01-01

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

Energy Technology Data Exchange (ETDEWEB)

Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

2006-03-30

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

ER-bound protein tyrosine phosphatase PTP1B interacts with Src at the plasma membrane/substrate interface.

Directory of Open Access Journals (Sweden)

Melisa C Monteleone

Full Text Available PTP1B is an endoplasmic reticulum (ER anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC. Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

Crystal structure of A. aeolicus LpxC with bound product suggests alternate deacetylation mechanism.

Science.gov (United States)

Miller, Matthew D; Gao, Ning; Ross, Philip L; Olivier, Nelson B

2015-09-01

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is the first committed step to form lipid A, an essential component of the outer membrane of Gram-negative bacteria. As it is essential for the survival of many pathogens, LpxC is an attractive target for antibacterial therapeutics. Herein, we report the product-bound co-crystal structure of LpxC from the acheal Aquifex aeolicus solved to 1.6 Å resolution. We identified interactions by hydroxyl and hydroxymethyl substituents of the product glucosamine ring that may enable new insights to exploit waters in the active site for structure-based design of LpxC inhibitors with novel scaffolds. By using this product structure, we have performed quantum mechanical modeling on the substrate in the active site. Based on our results and published experimental data, we propose a new mechanism that may lead to a better understanding of LpxC catalysis and inhibition. © 2015 Wiley Periodicals, Inc.

Occlusion of /sup 22/Na+ and /sup 86/Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

Energy Technology Data Exchange (ETDEWEB)

Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

1987-08-05

In this work, we examined occlusion of /sup 22/Na+ and /sup 86/Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of /sup 22/Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of /sup 22/Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by /sup 32/P incorporation from (gamma-/sup 32/P)CrATP. Maximum capacity for phosphorylation from (gamma-/sup 32/P)CrATP was 6 nmol/mg of protein and equal to capacities for binding of (48V)vanadate and (/sup 3/H)ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport.

A genetic screen for anchorage-independent proliferation in mammalian cells identifies a membrane-bound neuregulin.

Directory of Open Access Journals (Sweden)

Davide Danovi

2010-07-01

Full Text Available Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF, a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.

Cardiolipin effects on membrane structure and dynamics.

Science.gov (United States)

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

2013-12-23

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.

Pathogen-Specific Binding Soluble Down Syndrome Cell Adhesion Molecule (Dscam Regulates Phagocytosis via Membrane-Bound Dscam in Crab

Directory of Open Access Journals (Sweden)

Xue-Jie Li

2018-04-01

Full Text Available The Down syndrome cell adhesion molecule (Dscam gene is an extraordinary example of diversity that can produce thousands of isoforms and has so far been found only in insects and crustaceans. Cumulative evidence indicates that Dscam may contribute to the mechanistic foundations of specific immune responses in insects. However, the mechanism and functions of Dscam in relation to pathogens and immunity remain largely unknown. In this study, we identified the genome organization and alternative Dscam exons from Chinese mitten crab, Eriocheir sinensis. These variants, designated EsDscam, potentially produce 30,600 isoforms due to three alternatively spliced immunoglobulin (Ig domains and a transmembrane domain. EsDscam was significantly upregulated after bacterial challenge at both mRNA and protein levels. Moreover, bacterial specific EsDscam isoforms were found to bind specifically with the original bacteria to facilitate efficient clearance. Furthermore, bacteria-specific binding of soluble EsDscam via the complete Ig1–Ig4 domain significantly enhanced elimination of the original bacteria via phagocytosis by hemocytes; this function was abolished by partial Ig1–Ig4 domain truncation. Further studies showed that knockdown of membrane-bound EsDscam inhibited the ability of EsDscam with the same extracellular region to promote bacterial phagocytosis. Immunocytochemistry indicated colocalization of the soluble and membrane-bound forms of EsDscam at the hemocyte surface. Far-Western and coimmunoprecipitation assays demonstrated homotypic interactions between EsDscam isoforms. This study provides insights into a mechanism by which soluble Dscam regulates hemocyte phagocytosis via bacteria-specific binding and specific interactions with membrane-bound Dscam as a phagocytic receptor.

Crystal structure of the adenosine A _2A receptor bound to an antagonist reveals a potential allosteric pocket

Energy Technology Data Exchange (ETDEWEB)

Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K. (Stanford-MED); (ConfometRx); (UCB Pharma)

2017-02-06

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.

Single-particle tracking: applications to membrane dynamics.

Science.gov (United States)

Saxton, M J; Jacobson, K

1997-01-01

Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is observed, but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.

Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed

International Nuclear Information System (INIS)

Raap, J.; Hollander, J.; Ovchinnikova, T. V.; Swischeva, N. V.; Skladnev, D.; Kiihne, S.

2006-01-01

Interactions between 15 N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortunately, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in 13 C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C α signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the 31 P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The 13 C-peptide peaks were selectively detected in a 13 C-detected 1 H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The 13 C-MAOSS results thus independently confirms previous findings from 15 N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution 13 C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and

Instanton bound states in ABJM theory

Energy Technology Data Exchange (ETDEWEB)

Hatsuda, Yasuyuki [DESY Hamburg (Germany). Theory Group; Tokyo Institute of Technology (Japan). Dept. of Physics; Moriyama, Sanefumi [Nagoya Univ. (Japan). Kobayashi Maskawa Inst. and Graduate School of Mathematics; Okuyama, Kazumi [Shinshu Univ., Matsumoto, Nagano (Japan). Dept. of Physics

2013-06-15

The partition function of the ABJM theory receives non-perturbative corrections due to instanton effects. We study these non-perturbative corrections, including bound states of worldsheet instantons and membrane instantons, in the Fermi-gas approach. We require that the total non-perturbative correction should be always finite for arbitrary Chern-Simons level. This finiteness is realized quite non-trivially because each bound state contribution naively diverges at some levels. The poles of each contribution should be canceled out in total. We use this pole cancellation mechanism to find unknown bound state corrections from known ones. We conjecture a general expression of the bound state contribution. Summing up all the bound state contributions, we find that the effect of bound states is simply incorporated into the worldsheet instanton correction by a redefinition of the chemical potential in the Fermi-gas system. Analytic expressions of the 3- and 4-membrane instanton corrections are also proposed.

Hierarchically structured, nitrogen-doped carbon membranes

KAUST Repository

Wang, Hong

2017-08-03

The present invention is a structure, method of making and method of use for a novel macroscopic hierarchically structured, nitrogen-doped, nano-porous carbon membrane (HNDCMs) with asymmetric and hierarchical pore architecture that can be produced on a large-scale approach. The unique HNDCM holds great promise as components in separation and advanced carbon devices because they could offer unconventional fluidic transport phenomena on the nanoscale. Overall, the invention set forth herein covers a hierarchically structured, nitrogen-doped carbon membranes and methods of making and using such a membranes.

The ER in 3-D: a multifunctional dynamic membrane network

OpenAIRE

Friedman, Jonathan R.; Voeltz, Gia K.

2011-01-01

The endoplasmic reticulum (ER) is a large, singular, membrane-bound organelle that has an elaborate 3-D structure with a diversity of structural domains. It contains regions that are flat and cisternal, ones that are highly curved and tubular, and others adapted to form contact with nearly every other organelle and with the plasma membrane. ER 3-D structure is determined by both integral ER membrane proteins and by interactions with the cytoskeleton. Here, we describe some of the factors that...

UV inactivation of enzymes in supramolecular complexes of biological membranes. The phenomenon of photochemical allotropy

International Nuclear Information System (INIS)

Konev, S.V.; Volotovskij, I.D.; Sheiko, L.M.

1978-01-01

The photosensitivity of erythrocyte acetylcholinesterase (AChE) is different in its free and membrane-bound states. The modification of the structure of membraneous lipids by phospholipases A 2 , C and D or by cholesterol depletion is accompanied by a change in AChE photosensitivity. UV light was demonstrated to induce cooperative structural transitions in the erythrocyte membrane. This follows from the data obtained by circular dichroism and solubilization in detergents. In contrast to free AChE, UV light acts on the membraneous enzyme as a mixed inhibitor (simultaneous change in Vsub(max) and Ksub(m)). The anomalous behaviour of membrane-bound enzyme, termed the phenomenon of photochemical allotropy, is associated with a modification of the structure within the microenvironment of the residual AChE. The phenomenon depends on membrane integrity, and disappears after treatment of erythrocyte ghosts with ultrasound, trypsin, phospholipases and neuraminidase and remains unchanged in cholesterol-depleted membranes. The nature and localization of events responsible for this phenomenon are discussed. (author)

Self-Assembling Peptide Surfactants A6K and A6D Adopt a-Helical Structures Useful for Membrane Protein Stabilization

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Furen Zhuang

2011-10-01

Full Text Available Elucidation of membrane protein structures have been greatly hampered by difficulties in producing adequately large quantities of the functional protein and stabilizing them. A6D and A6K are promising solutions to the problem and have recently been used for the rapid production of membrane-bound G protein-coupled receptors (GPCRs. We propose that despite their short lengths, these peptides can adopt α-helical structures through interactions with micelles formed by the peptides themselves. These α-helices are then able to stabilize α-helical motifs which many membrane proteins contain. We also show that A6D and A6K can form β-sheets and appear as weak hydrogels at sufficiently high concentrations. Furthermore, A6D and A6K together in sodium dodecyl sulfate (SDS can form expected β-sheet structures via a surprising α-helical intermediate.

How membrane lipids control the 3D structure and function of receptors

OpenAIRE

Jacques Fantini; Francisco J. Barrantes

2018-01-01

The cohabitation of lipids and proteins in the plasma membrane of mammalian cells is controlled by specific biochemical and biophysical rules. Lipids may be either constitutively tightly bound to cell-surface receptors (non-annular lipids) or less tightly attached to the external surface of the protein (annular lipids). The latter are exchangeable with surrounding bulk membrane lipids on a faster time scale than that of non-annular lipids. Not only do non-annular lipids bind to membrane prote...

Removal of Bound Triton X-100 from Purified Bovine Heart Cytochrome bc1

OpenAIRE

Varhač, Rastislav; Robinson, Neal C.; Musatov, Andrej

2009-01-01

Cytochrome bc1 isolated from Triton X-100 solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nmol of the enzyme. Purified cytochrome bc1 is fully active; however, protein bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc1 was accomplished by incubation with Bio-Beads SM-2 in presence of sodium cholate. Sodium cholate is critical since it does not interfere with the adsorpt...

Relationship of membrane-bound sulfhydryl groups to vitamin D-stimulated uptake of [75Se]Selenite by the brush border membrane vesicles from chick duodenum

International Nuclear Information System (INIS)

Mykkanen, H.M.; Wasserman, R.H.

1990-01-01

The uptake of selenite by purified brush border membrane vesicles isolated from duodena of rachitic or vitamin D-treated chicks was studied by using radioactive selenite and a rapid filtration technique. Cholecalciferol treatment (500 IU at 72 h) significantly enhanced selenite uptake, a response that decreased when the vesicles were stored at room temperature for 2.5 h prior to the uptake measurement. Preincubation of the vesicles in 1.0 mmol/L H2O2 reduced [75Se]selenite uptake, indicating the involvement of oxidizable groups in the uptake reaction. Iodoacetic acid (IAA), a sulfhydryl-blocking reagent, at 1-2 mmol/L concentration eliminated the difference in selenite uptake due to cholecalciferol and had no effect on vesicles from rachitic animals. A higher concentration of IAA (10 mmol/L) enhanced selenite uptake manyfold and increased the absolute difference due to cholecalciferol treatment. Single intravenous doses of 100 IU cholecalciferol, 100 IU ergocalciferol, or 0.1 micrograms 1,25-dihydroxycholecalciferol also stimulated selenite uptake, suggesting a general response to vitamin D compounds. Normal animals given a single dose of 1,25-dihydroxycholecalciferol 12 h prior to killing also responded. Treatments that enhanced the uptake of [75Se]selenite also increased the amount of membrane-bound sulfhydryl groups, suggesting the involvement of membrane-bound sulfhydryl groups in the vitamin D response. A significant increase in selenite uptake by intravenous 1,25-dihydroxycholecalciferol occurred within 10 min. This rapid effect provides a new tool to probe early biochemical effects of vitamin D on intestinal epithelium

Static and Dynamic Membrane Structures

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Sergiu Ivanov

2012-10-01

Full Text Available While originally P systems were defined to contain multiset rewriting rules, it turned out that considering different types of rules may produce important results, such as increasing the computational power of the rules. This paper focuses on factoring out the concept of a membrane structure out of various P system models with the goal of providing useful formalisations. Both static and dynamic membrane structures are considered.

Coherent structures in wall-bounded turbulence

Science.gov (United States)

Jiménez, Javier

2018-05-01

This article discusses the description of wall-bounded turbulence as a deterministic high-dimensional dynamical system of interacting coherent structures, defined as eddies with enough internal dynamics to behave relatively autonomously from any remaining incoherent part of the flow. The guiding principle is that randomness is not a property, but a methodological choice of what to ignore in the flow, and that a complete understanding of turbulence, including the possibility of control, requires that it be kept to a minimum. After briefly reviewing the underlying low-order statistics of flows at moderate Reynolds numbers, the article examines what two-point statistics imply for the decomposition of the flow into individual eddies. Intense eddies are examined next, including their temporal evolution, and shown to satisfy many of the properties required for coherence. In particular, it is shown that coherent structures larger than the Corrsin scale are a natural consequence of the shear. In wall-bounded turbulence, they can be classified into coherent dispersive waves and transient bursts. The former are found in the viscous layer near the wall and as very-large structures spanning the boundary layer thickness. Although they are shear-driven, these waves have enough internal structure to maintain a uniform advection velocity. Conversely, bursts exist at all scales, are characteristic of the logarithmic layer, and interact almost linearly with the shear. While the waves require a wall to determine their length scale, the bursts are essentially independent from it. The article concludes with a brief review of our present theoretical understanding of turbulent structures, and with a list of open problems and future perspectives.

Protective Effect of Prosopis cineraria Against N-Nitrosodiethylamine Induced Liver Tumor by Modulating Membrane Bound Enzymes and Glycoproteins

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Naina Mohamed Pakkir Maideen

2012-06-01

Full Text Available Purpose: The objective of the present study was to evaluate the protective effect of methanol extract of Prosopis cineraria (MPC against N-nitrosodiethylamine (DEN, 200mg/kg induced Phenobarbital promoted experimental liver tumors in male Wistar rats. Methods: The rats were divided into four groups, each group consisting of six animals. Group 1 served as control animals. Liver tumor was induced in group 2, 3, and 4 and Group 3 animals received MPC 200mg/kg and Group 4 animals received MPC 400mg/kg. Results: Administration of DEN has brought down the levels of membrane bound enzymes like Na+/ K+ ATPase, Mg2+ ATPase and Ca2+ATPase which were later found to be increased by the administration of Prosopis cineraria (200 and 400mg/kg in dose dependent manner. The MPC extract also suppressed the levels of glycoproteins like Hexose, Hexosamine and Sialic acid when compared to liver tumor bearing animals. Conclusions: Our study suggests that MPC may extend its protective role by modulating the levels of membrane bound enzymes and suppressing glycoprotein levels.

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

DEFF Research Database (Denmark)

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

2016-01-01

Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid...... compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have...... the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic...

Mobility of drugs in lipid membranes by NMR

International Nuclear Information System (INIS)

Yoshii, Noriyuki; Okamura, Emiko

2011-01-01

Mobility of drugs and biomembrane constituents is a key to elucidate the membrane transport mechanism in the cell. Lipid bilayer membrane is a dynamic structure where molecules are always fluctuating under physiological conditions. The mechanism of drug transport is related to the molecular dynamics in such soft, fluid membrane interface. To gain insight into molecular movements in membranes, we develop a noninvasive method to monitor dynamics properties of drugs and lipid components in membranes by applying multinuclear high-resolution solution NMR in combination with the pulsed-field-gradient (PFG) technique. We have quantified the diffusivity, the kinetics of membrane binding, and the bound fraction of the drug in situ by using large unilamellar vesicles of egg phosphatidylcholine as model cell membranes. The combination of 1D and PFG NMR serves to quantify the kinetics of membrane binding where the bound and the free components are unable to distinguish because of the rapid exchange on the NMR timescale. A small-sized 5-fluorouracil and fluorinated bisphenol A are used as model drug. (author)

Graphene-based structure, method of suspending graphene membrane, and method of depositing material onto graphene membrane

Science.gov (United States)

Zettl, Alexander K.; Meyer, Jannik Christian

2013-04-02

An embodiment of a method of suspending a graphene membrane across a gap in a support structure includes attaching graphene to a substrate. A pre-fabricated support structure having the gap is attached to the graphene. The graphene and the pre-fabricated support structure are then separated from the substrate which leaves the graphene membrane suspended across the gap in the pre-fabricated support structure. An embodiment of a method of depositing material includes placing a support structure having a graphene membrane suspended across a gap under vacuum. A precursor is adsorbed to a surface of the graphene membrane. A portion of the graphene membrane is exposed to a focused electron beam which deposits a material from the precursor onto the graphene membrane. An embodiment of a graphene-based structure includes a support structure having a gap, a graphene membrane suspended across the gap, and a material deposited in a pattern on the graphene membrane.

Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

2010-11-03

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase.

Science.gov (United States)

Ahrens, M L

1981-04-06

In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.

The ER in 3D: a multifunctional dynamic membrane network.

Science.gov (United States)

Friedman, Jonathan R; Voeltz, Gia K

2011-12-01

The endoplasmic reticulum (ER) is a large, singular, membrane-bound organelle that has an elaborate 3D structure with a diversity of structural domains. It contains regions that are flat and cisternal, ones that are highly curved and tubular, and others adapted to form contacts with nearly every other organelle and with the plasma membrane. The 3D structure of the ER is determined by both integral ER membrane proteins and by interactions with the cytoskeleton. In this review, we describe some of the factors that are known to regulate ER structure and discuss how this structural organization and the dynamic nature of the ER membrane network allow it to perform its many different functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Overcoming barriers to membrane protein structure determination.

Science.gov (United States)

Bill, Roslyn M; Henderson, Peter J F; Iwata, So; Kunji, Edmund R S; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G; Vogel, Horst

2011-04-01

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

Science.gov (United States)

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

The effect of near-infrared MLS laser radiation on cell membrane structure and radical generation.

Science.gov (United States)

Kujawa, Jolanta; Pasternak, Kamila; Zavodnik, Ilya; Irzmański, Robert; Wróbel, Dominika; Bryszewska, Maria

2014-09-01

The therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the biochemical mechanism of the interaction of laser light with living cells is not fully understood. We have investigated the effect of MLS (Multiwave Locked System) laser near-infrared irradiation on cell membrane structure, functional properties, and free radical generation using human red blood cells and breast cancer MCF-4 cells. The cells were irradiated with low-intensity MLS near-infrared (simultaneously 808 nm, continuous emission and 905 nm, pulse emission, pulse-wave frequency, 1,000 or 2,000 Hz) laser light at light doses from 0 to 15 J (average power density 212.5 mW/cm(2), spot size was 3.18 cm(2)) at 22 °C, the activity membrane bound acetylcholinesterase, cell stability, anti-oxidative activity, and free radical generation were the parameters used in characterizing the structural and functional changes of the cell. Near-infrared low-intensity laser radiation changed the acetylcholinesterase activity of the red blood cell membrane in a dose-dependent manner: There was a considerable increase of maximal enzymatic rate and Michaelis constant due to changes in the membrane structure. Integral parameters such as erythrocyte stability, membrane lipid peroxidation, or methemoglobin levels remained unchanged. Anti-oxidative capacity of the red blood cells increased after MLS laser irradiation. This irradiation induced a time-dependent increase in free radical generation in MCF-4 cells. Low-intensity near-infrared MLS laser radiation induces free radical generation and changes enzymatic and anti-oxidative activities of cellular components. Free radical generation may be the mechanism of the biomodulative effect of laser radiation.

Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state.

Science.gov (United States)

Kanai, Ryuta; Ogawa, Haruo; Vilsen, Bente; Cornelius, Flemming; Toyoshima, Chikashi

2013-10-10

Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C

International Nuclear Information System (INIS)

Lapetina, E.G.; Reep, B.R.

1987-01-01

We have assessed the binding of [alpha- 32 P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO 4 /PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha- 32 P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha- 32 P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S

Poly(acrylonitrile)chitosan composite membranes for urease immobilization.

Science.gov (United States)

Gabrovska, Katya; Georgieva, Aneliya; Godjevargova, Tzonka; Stoilova, Olya; Manolova, Nevena

2007-05-10

(Poly)acrylonitrile/chitosan (PANCHI) composite membranes were prepared. The chitosan layer was deposited on the surface as well as on the pore walls of the base membrane. This resulted in the reduction of the pore size of the membrane and in an increase of their hydrophilicity. The pore structure of PAN and PANCHI membranes were determined by TEM and SEM analyses. It was found that the average size of the pore under a selective layer base PAN membrane is 7 microm, while the membrane coated with 0.25% chitosan shows a reduced pore size--small or equal to 5 microm and with 0.35% chitosan--about 4 microm. The amounts of the functional groups, the degree of hydrophilicity and transport characteristics of PAN/Chitosan composite membranes were determined. Urease was covalently immobilized onto all kinds of PAN/chitosan composite membranes using glutaraldehyde. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity (94%) was measured for urease bound to PANCHI2 membranes (0.25% chitosan). The basic characteristics (pH(opt), pH(stability), T(opt), T(stability), heat inactivation and storage stability) of immobilized urease were determined. The obtained results show that the poly(acrylonitrile)chitosan composite membranes are suitable for enzyme immobilization.

Role for chlamydial inclusion membrane proteins in inclusion membrane structure and biogenesis.

Directory of Open Access Journals (Sweden)

Jeffrey Mital

Full Text Available The chlamydial inclusion membrane is extensively modified by the insertion of type III secreted effector proteins. These inclusion membrane proteins (Incs are exposed to the cytosol and share a common structural feature of a long, bi-lobed hydrophobic domain but little or no primary amino acid sequence similarity. Based upon secondary structural predictions, over 50 putative inclusion membrane proteins have been identified in Chlamydia trachomatis. Only a limited number of biological functions have been defined and these are not shared between chlamydial species. Here we have ectopically expressed several C. trachomatis Incs in HeLa cells and find that they induce the formation of morphologically distinct membranous vesicular compartments. Formation of these vesicles requires the bi-lobed hydrophobic domain as a minimum. No markers for various cellular organelles were observed in association with these vesicles. Lipid probes were incorporated by the Inc-induced vesicles although the lipids incorporated were dependent upon the specific Inc expressed. Co-expression of Inc pairs indicated that some colocalized in the same vesicle, others partially overlapped, and others did not associate at all. Overall, it appears that Incs may have an intrinsic ability to induce membrane formation and that individual Incs can induce membranous structures with unique properties.

Integral membrane protein structure determination using pseudocontact shifts

Energy Technology Data Exchange (ETDEWEB)

Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

2015-04-15

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

Membrane-bound alcohol dehydrogenase is essential for glyceric acid production in Acetobacter tropicalis.

Science.gov (United States)

Habe, Hiroshi; Sato, Shun; Fukuoka, Tokuma; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

2011-01-01

Acetobacter tropicalis NBRC16470 can produce highly enantiomerically pure D-glyceric acid (D-GA; >99 % enantiomeric excess) from glycerol. To investigate whether membrane-bound alcohol dehydrogenase (mADH) is involved in GA production in A. tropicalis, we amplified part of the gene encoding mADH subunit I (adhA) using polymerase chain reaction and constructed an adhA-disrupted mutant of A. tropicalis (ΔadhA). Because ΔadhA did not produce GA, we confirmed that mADH is essential for the conversion of glycerol to GA. We also cloned and sequenced the entire region corresponding to adhA and adhB, which encodes mADH subunit II. The sequences showed high identities (84-86 %) with the equivalent mADH subunits from other Acetobacter spp.

Wrinkling reduction of membrane structure by trimming edges

Directory of Open Access Journals (Sweden)

Mingjun Liu

2017-05-01

Full Text Available Thin membranes have negligible bending stiffness, compressive stresses inevitably lead to wrinkling. Therefore, it is important to keep the surface of membrane structures flat in order to guarantee high precision. Edge-trimming is an effective method to passively diminish wrinkles, however a key difficulty in this process is the determination of the optimal trimming level. In this paper, regular polygonal membrane structures subjected to equal radial forces were analyzed, and a new stress field distribution model for arc-edge square membrane structure was proposed to predict the optimal trimming level. This model is simple and applicable to any polygonal membrane structures. Comparison among the results of the finite element analysis, and the experimental and analytical results showed that the proposed model accurately described the stress field distribution and guaranteed that there are no wrinkles appear inside the effective inscribed circle region for the optimal trimming level.

Characterization of soluble and membrane-bound alkaline phosphatase in Nilaparvata lugens and their potential relation to development and insecticide resistance.

Science.gov (United States)

Wang, Zengxia; Liu, Shuhua; Yang, Baojun; Liu, Zewen

2011-09-01

Two forms (soluble and membrane-bound) of alkaline phosphatases (ALPs) were found in the brown planthopper, Nilaparvata lugens. In order to further study ALPs in N. lugens, two putative ALP genes (Nl-ALP1 and Nl-ALP2) were identified in this pest. Both Nl-ALP1 and Nl-ALP2 show approximately the same degree of sequence identity (40-50%) to other insect soluble and membrane-bound forms of ALP. Correlation of ALP activity and mRNA levels at different developmental stages, or following application of 20-hydroxyecdysone (20E) and insecticide fenvalerate, suggests that Nl-ALP1 and Nl-ALP2 might encode a soluble (sALP) and a membrane-bound ALP (mALP), respectively. Nl-ALP1-specific antibody Nl1-I detected only a specific band in soluble protein preparations and Nl-ALP2 specific antibody Nl2-I only detected a specific band in insoluble protein preparations, which provided conclusive linkages between Nl-ALP1 and a sALP and between Nl-ALP2 and a m ALP. Then, Nl-ALP1 was denoted as Nl-sALP for a sALP and Nl-ALP2 was denoted as Nl-mALP for a mALP. Only sALP activity and Nl-sALP mRNA level were induced by 20E and fenvalerate, which was confirmed by the density of specific band detected by Nl1-I in Sus strain with or without fenvalerate treatment. Additionally, the sALP activity, as well as Nl-sALP mRNA level, was significantly higher in a fenvalerate resistant population, compared with Sus strain. These results indicate that the sALP is more responsive to chemical stimulus, such as hormone and insecticide, and might play dual roles in development and insecticide tolerance. © 2011 Wiley Periodicals, Inc.

Studying Membrane Protein Structure and Function Using Nanodiscs

DEFF Research Database (Denmark)

Huda, Pie

The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important...

An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

Energy Technology Data Exchange (ETDEWEB)

Walker, C.J.; Weinstein, J.D. (Clemson Univ, SC (United States))

1991-05-01

Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

Science.gov (United States)

Kupisz, Kamila; Sujak, Agnieszka; Patyra, Magdalena; Trebacz, Kazimierz; Gruszecki, Wiesław I

2008-10-01

Polar carotenoid pigment zeaxanthin (beta,beta-carotene-3,3'-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 x 10(7) Omega cm2 (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H+-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21 degrees with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of beta-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.

Effect of dope solution temperature on the membrane structure and membrane distillation performance

Science.gov (United States)

Nawi, N. I. M.; Bilad, M. R.; Nordin, N. A. H. M.

2018-04-01

Membrane distillation (MD) is a non-isothermal process applicable to purify water using hydrophobic membrane. Membrane in MD is hydrophobic, permeable to water vapor but repels liquid water. MD membrane is expected to pose high flux, high fouling and scaling resistances and most importantly high wetting resistance. This study develops flat-sheet polyvinylidene fluoride (PVDF) membrane by exploring both liquid-liquid and liquid-solid phase inversion technique largely to improve its wetting resistance and flux performance. We hypothesize that temperature of dope solution play roles in solid-liquid separation during membrane formation and an optimum balance between liquid-liquid and liquid-solid (crystallization) separation leads to highly performance PVDF membrane. Findings obtained from differential scanning calorimeter test show that increasing dope solution temperature reduces degree of PVDF crystallinity and suppresses formation of crystalline structure. The morphological images of the resulting membranes show that at elevated dope solution temperature (40, 60, 80 and 100°C), the spherulite-like structures are formed across the thickness of membranes ascribed from due to different type of crystals. The performance of direct-contact MD shows that the obtained flux of the optimum dope temperature (60°C) of 10.8 L/m2h is comparable to commercial PTFE-based MD membrane.

New low-flux mixed matrix membranes that offer superior removal of protein-bound toxins from human plasma

Science.gov (United States)

Pavlenko, Denys; van Geffen, Esmée; van Steenbergen, Mies J.; Glorieux, Griet; Vanholder, Raymond; Gerritsen, Karin G. F.; Stamatialis, Dimitrios

2016-10-01

Hemodialysis is a widely available and well-established treatment for patients with End Stage Renal Disease (ESRD). However, although life-sustaining, patient mortality rates are very high. Several recent studies corroborated the link between dialysis patients’ outcomes and elevated levels of protein-bound uremic toxins (PBUT) that are poorly removed by conventional hemodialysis. Therefore, new treatments are needed to improve their removal. Recently, our group showed that the combination of dialysis and adsorption on one membrane, the mixed matrix membrane (MMM), can effectively remove those toxins from human plasma. However, these first MMMs were rather large in diameter and their mass transport characteristics needed improvement before application in the clinical setting. Therefore, in this study we developed a new generation of MMMs that have a smaller diameter and optimized characteristics offering superior ability in removing the PBUT indoxyl sulfate (IS) and p-cresyl sulfate (pCS) in comparison to first generation MMMs (30 and 125% respectively), as well as, a commercial dialysis membrane (more than 100% better removal).

Effect of saline stress on plasma membrane structure and function of barley roots

International Nuclear Information System (INIS)

Rahmani, F. H.

2000-01-01

Barely (Hordeum vulgare L. c v. Black Local) plants were grown hydroponic ally under different saline stresses (50, 100, 150 And 200 mm NaCI. The adverse effect of each saline stress on the structure and function of root cells plasma membrane was studied in terms of root surface ATPase activation by NaCI in the reaction mixture. Was 0, 50, 100. 150 and 200mM. ATPase activity was found to be increased gradually at certain concentrations of NaCI. For control and 50mM stressed plants, the increase in root surface ATPase activity was started at 150mM NaCI. For 100mM stressed plants it was started at 100mM NaCI. For 150 and 200mM stressed plants it was stated at 50mM NaCI Results indicated that the adverse effect of the growth medium saline stresses on the integrity of the plasma membrane was started at 100mM saline stress. Accordingly the role of plasma membrane bound ATPase in active ion transport was disturbed at 100mM saline stress and may be impaired at 150 and 200mM saline stresses. It was suggested that the lipid environment of the plasma membrane surrounding ATPase was modified by the saline stresses 100-200mM. (author). 38 refs., 2 figs., 2 tabs

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

Science.gov (United States)

Merz, Frieder; Boehringer, Daniel; Schaffitzel, Christiane; Preissler, Steffen; Hoffmann, Anja; Maier, Timm; Rutkowska, Anna; Lozza, Jasmin; Ban, Nenad; Bukau, Bernd; Deuerling, Elke

2008-01-01

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome–nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors. PMID:18497744

Differential expression of growth factor receptors and membrane-bound tumor markers for imaging in male and female breast cancer.

Directory of Open Access Journals (Sweden)

Jeroen F Vermeulen

Full Text Available INTRODUCTION: Male breast cancer accounts for 0.5-1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer. METHODS: Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers. RESULTS: Growth factor receptors were variably expressed in 4.5% (MET up to 38.5% (IGF1-R of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers. CONCLUSIONS: Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of

Membrane-assisted growth of DNA origami nanostructure arrays.

Science.gov (United States)

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

2015-01-01

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

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

Directory of Open Access Journals (Sweden)

Sandy Azzi

2015-06-01

Full Text Available Intrarenal interleukin-15 (IL-15 participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15 isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC to peritumoral (ptumTEC, tumoral (RCC, and cancer stem cells (CSC/CD105+. RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15 isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα. This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

Electrospun superhydrophobic membranes with unique structures for membrane distillation.

Science.gov (United States)

Liao, Yuan; Loh, Chun-Heng; Wang, Rong; Fane, Anthony G

2014-09-24

With modest temperature demand, low operating pressure, and high solute rejection, membrane distillation (MD) is an attractive option for desalination, waste treatment, and food and pharmaceutical processing. However, large-scale practical applications of MD are still hindered by the absence of effective membranes with high hydrophobicity, high porosity, and adequate mechanical strength, which are important properties for MD permeation fluxes, stable long-term performance, and effective packing in modules without damage. This study describes novel design strategies for highly robust superhydrophobic dual-layer membranes for MD via electrospinning. One of the newly developed membranes comprises a durable and ultrathin 3-dimensional (3D) superhydrophobic skin and porous nanofibrous support whereas another was fabricated by electrospinning 3D superhydrophobic layers on a nonwoven support. These membranes exhibit superhydrophobicity toward distilled water, salty water, oil-in-water emulsion, and beverages, which enables them to be used not only for desalination but also for other processes. The superhydrophobic dual-layer membrane #3S-N with nanofibrous support has a competitive permeation flux of 24.6 ± 1.2 kg m(-2) h(-1) in MD (feed and permeate temperate were set as 333 and 293 K, respectively) due to the higher porosity of the nanofibrous scaffold. Meanwhile, the membranes with the nonwoven support exhibit greater mechanical strength due to this support combined with better long-term performance because of the thicker 3D superhydrophobic layers. The morphology, pore size, porosity, mechanical properties, and liquid enter pressure of water of these superhydrophobic composite membranes with two different structures are reported and compared with commercial polyvinylidene fluoride membranes.

Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

Science.gov (United States)

Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

2016-04-25

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

Science.gov (United States)

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

The limits of bound structures in the accelerating Universe

NARCIS (Netherlands)

Dunner, R; Araya, PA; Meza, A; Reisenegger, A

2006-01-01

According to the latest evidence, the Universe is entering an era of exponential expansion, where gravitationally bound structures will get disconnected from each other, forming isolated 'island universes'. In this scenario, we present a theoretical criterion to determine the boundaries of

Loss of covalently linked lipid as the mechanism for radiation-induced release of membrane-bound polysaccharide and exonuclease from Micrococcus radiodurans

International Nuclear Information System (INIS)

Mitchel, R.E.J.

1981-01-01

The mechanism of γ-radiation-induced release of polysaccharide and exonuclease from the midwall membrane of Micrococcus radiodurans has been examined. These two components appear to be released independently, but by very similar processes. Direct analysis of radiation-released polysaccharide indicated the absence of an alkali-labile neutral lipid normally present in the native material. Radiation-induced release therefore probably results from the radiolytic cleavage of a covalently linked lipid which normally serves to anchor these substances to the membrane. The absence of a natural membrane-bound carotenoid had no effect on the rate of release of these components. Likewise, the absence of exonuclease in an exonuclease minus mutant did not influence the release of polysaccharide. It is suggested that the major pathway of radical transfer from the initiating .OH and culminating in the cleavage of the neutral lipid anchor may not be via the membrane

Nonreutilizaton of adrenal chromaffin granule membranes following secretion

International Nuclear Information System (INIS)

Nobiletti, J.B.

1985-01-01

The intracellular postexocytotic fate of the adrenal chromaffin granule membrane (reutilization vs. nonreutilization) was addressed through two experimental approaches. First, ( 3 H) leucine pulse-chase labeling experiments were conducted in two systems - the isolated retrograde perfused cat adrenal gland and cultured bovine adrenal chromaffin cells to compare chromaffin granule soluble dopamine-B-hydroxylase (DBH) turnover (marker for granule soluble content turnover) to that of membrane-bound DBH (marker for granule membrane turnover). Experiments in cat adrenal glands showed that at all chase periods the granule distribution of radiolabeled DBH was in agreement with the DBH activity distribution (73% membrane-bound/27% soluble) - a result consistent with parallel turnover of soluble and membrane-bound DBH. Experiments in cultured bovine cells showed that labeled soluble and membrane-bound DBH had parallel turnover patterns and at all chase period, the distribution of radiolabeled DBH between the soluble contents and membranes was similar to the DBH activity distribution (50% soluble/50% membrane-bound). The above experiments showed that the soluble contents and membranes turnover in parallel and are consistent with nonreutilization of chromaffin granule membranes following exocytosis. Isolated retrograde perfused bovine adrenal glands were subjected to repetitive acetylcholine stimulation to induce exocytosis and then the dense and less-dense chromaffin granule fractions were isolated. Since both approaches gave results consistent with membrane nonreutilization, the authors conclude that once a chromaffin granule is involved in exocytosis, its membrane is not reutilized for the further synthesis, storage, and secretion of catecholamines

Investigating membrane-bound Argonaute functions in Arabidopsis

DEFF Research Database (Denmark)

Barghetti, Andrea

and how AGO1 membrane recruitment is mediated as well as its functional importance remain poorly characterized. Isoprenoid biogenesis was previously found to be required for both AGO1 activity and membrane association, but the mechanistic connection between the two pathways was not discovered. Since....... The key effectors of sRNA-guided gene regulation are ARGONAUTE (AGO) proteins. A group of Heat Shock Proteins of the HSP70/HSP90 chaperone machinery mediates the process, termed loading, that allow the functional association of sRNA with AGOs. Upon loading, Argonautes regulate complementary mRNA targets...... with the rough endoplasmic reticulum (rER). Membranelocalized argonaute functions include translational repression, production of secondary phased small interfering RNA (siRNA) and autophagy-mediated turnover. However proteins interacting with AGO1 specifically on membrane fractions have not been identified...

Crystals of Na(+)/K(+)-ATPase with bound cisplatin.

Science.gov (United States)

Huliciak, Miroslav; Reinhard, Linda; Laursen, Mette; Fedosova, Natalya; Nissen, Poul; Kubala, Martin

2014-12-01

Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na(+)/K(+)-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4 Å and 7.9 Å), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal.

Science.gov (United States)

Nossoni, Zahra; Assar, Zahra; Yapici, Ipek; Nosrati, Meisam; Wang, Wenjing; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James

2014-12-01

Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.

Vulnerable Derivatives and Good Deal Bounds: A Structural Model

DEFF Research Database (Denmark)

Murgoci, Agatha

2013-01-01

We price vulnerable derivatives -- i.e. derivatives where the counterparty may default. These are basically the derivatives traded on the over-the-counter (OTC) markets. Default is modeled in a structural framework. The technique employed for pricing is good deal bounds (GDBs). The method imposes...

Deformation analysis of a film-overlapped micro-pump membrane structure

International Nuclear Information System (INIS)

Lee, Fu-Shin; Wang, Pi-Wen; Chen, Chih-Hsiung

2008-01-01

A novel approach is developed to study a film-overlapped membrane structure. Meanwhile, the established model is employed to design the micro-pump membrane structure and to evaluate its pumping efficiency. Two-dimensional coupling effects between the overlapping actuator films and the deformable membrane are thoroughly investigated, including the influences on the membrane from the overlapping films' elongation effects, Poisson's ratio effects and shear strain effects. Overall deformations and interactions for the three-layer membrane structures are accurately calculated through exercising the developed model, in contrast to what difficulties are usually encountered in carrying out FEM methods with very thin elements meshed for the actuator films. Furthermore, this study demonstrates that the high stiffness of the actuating metal films needs to be reflected in the equivalent stiffness of the membrane structures, especially when the sizes of the actuator films become compatible with the sizes of the membranes. Hence, the optimal micro-pumping efficiency of a membrane structure is acquired upon exercising the developed model, and larger sizes of the actuating films do not definitely obtain larger pumping efficiencies for the electromagnetically actuated micro-pumps

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

International Nuclear Information System (INIS)

Terawaki, Shin-ichi; Kitano, Ken; Aoyama, Miki; Hakoshima, Toshio

2008-01-01

The radixin FERM domain was shown to bind the MT1-MMP cytoplasmic peptide and crystals of the complex were obtained. ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6 1 22, with unit-cell parameters a = b = 122.7, c = 128.3 Å, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 Å

Structure and hydration of membranes embedded with voltage-sensing domains.

Science.gov (United States)

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

2009-11-26

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

Preliminary Results on the Experimental Investigation of the Structure Functions of Bound Nucleons

Energy Technology Data Exchange (ETDEWEB)

Bodek, Arie [Univ. of Rochester, NY (United States); Thomas Jefferson National Accelerator Facility (TJNAF), Newport News, VA (United States)

2016-08-01

We present preliminary results on an experimental study of the nuclear modification of the longitudinal ($\\sigma_L$) and transverse ($\\sigma_T$) structure functions of nucleons bound in nuclear targets. The origin of these modifications (commonly referred as as the EMC effect) is not fully understood. Our measurements of R= $\\sigma_L / \\sigma_T$ for nuclei ($R_A$) and for deuterium ($R_D$) indicate that nuclear modifications of the structure functions of bound nucleons are different for the longitudinal and transverse structure functions, and that contrary to expectation from several theoretical models, $R_A< R_D$.

Membrane-Assisted Growth of DNA Origami Nanostructure Arrays

Science.gov (United States)

2015-01-01

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

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

International Nuclear Information System (INIS)

Chiu, T.H.; Yu, Onnfoh; Rosenberg, H.C.

1989-01-01

Irreversible incorporation of [ 3 H]flunitrazepam and [ 3 H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV/irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [ 3 H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [ 3 H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [ 3 H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [ 3 H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [ 3 H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex

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

International Nuclear Information System (INIS)

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

2015-01-01

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

Hierarchically structured, nitrogen-doped carbon membranes

KAUST Repository

Wang, Hong; Wu, Tao

2017-01-01

The present invention is a structure, method of making and method of use for a novel macroscopic hierarchically structured, nitrogen-doped, nano-porous carbon membrane (HNDCMs) with asymmetric and hierarchical pore architecture that can be produced

USE OF BOUNDING ANALYSES TO ESTIMATE THE PREFORMANCE OF A SEISMICALLY ISOLATED STRUCTURE

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Gökhan ÖZDEMİR

2017-03-01

Full Text Available Current design approach for seismic isolated structures is to perform bounding analyses. These analyses provide an envelope for the response of the seismic isolated structure rather than focusing on the actual performance. In this study, the success of bounding analyses to estimate performance of a seismic isolated structure, in which the isolation is provided by means of lead rubber bearings (LRBs, is evaluated in a comparative manner. For this purpose, nonlinear response history analyses were performed under the effect of bidirectional ground motion excitations. In bounding analyses, non-deteriorating hysteretic representations were used to model the hysteretic behavior of LRBs. On the other hand, to estimate the actual performance of both the superstructure and isolator units, deteriorating hysteretic idealizations were employed. The deterioration in strength of LRBs was defined as a function of temperature rise in the lead core. The analyzed structure is an existing seismically isolated hospital building and analytically modeled in accordance with its reported design properties for both isolation units and superstructure. Results obtained from analyses where LRBs are idealized by both deteriorating and non-deteriorating hysteretic representations are used in the comparisons. The response quantities used in the comparisons are maximum isolator displacement, maximum isolator force, maximum absolute floor acceleration, and maximum relative story displacements. In an average sense, bounding analyses is found to provide conservative estimates for the selected response quantities and fulfills its intended purpose. However, it is revealed that there may be individual cases where bounding analyses fails to provide a safe envelope.

Flow-alignment of bicellar lipid mixtures: orientations of probe molecules and membrane-associated biomacromolecules in lipid membranes studied with polarized light

KAUST Repository

Kogan, Maxim; Beke-Somfai, Tamá s; Nordé n, Bengt

2011-01-01

Bicelles are excellent membrane-mimicking hosts for a dynamic and structural study of solutes with NMR, but the magnetic fields required for their alignment are hard to apply to optical conditions. Here we demonstrate that bicellar mixtures can be aligned by shear forces in a Couette flow cell, to provide orientation of membrane-bound retinoic acid, pyrene and cytochrome c (cyt c) protein, conveniently studied with linear dichroism spectroscopy. © 2011 The Royal Society of Chemistry.

Enhanced water desalination performance through hierarchically-structured ceramic membranes

NARCIS (Netherlands)

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

2017-01-01

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

Influence of fermentation liquid from waste activated sludge on anoxic/oxic- membrane bioreactor performance: Nitrogen removal, membrane fouling and microbial community.

Science.gov (United States)

Han, Xiaomeng; Zhou, Zhen; Mei, Xiaojie; Ma, Yan; Xie, Zhenfang

2018-02-01

In order to investigate effects of waste activated sludge (WAS) fermentation liquid on anoxic/oxic- membrane bioreactor (A/O-MBR), two A/O-MBRs with and without WAS fermentation liquid addition were operated in parallel. Results show that addition of WAS fermentation liquid clearly improved denitrification efficiency without deterioration of nitrification, while severe membrane fouling occurred. WAS fermentation liquid resulted in an elevated production of proteins and humic acids in bound extracellular polymeric substance (EPS) and release of organic matter with high MW fractions in soluble microbial product (SMP) and loosely bound EPS (LB-EPS). Measurement of deposition rate and fluid structure confirmed increased fouling potential of SMP and LB-EPS. γ-Proteobacteria and Ferruginibacter, which can secrete and export EPS, were also found to be abundant in the MBR with WAS fermentation liquid. It is implied that when WAS fermentation liquid was applied, some operational steps to control membrane fouling should be employed. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2016-03-01

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

Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA

Directory of Open Access Journals (Sweden)

Yijin Liu

2015-12-01

Full Text Available We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction.

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

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Hao Yang

2018-01-01

Full Text Available Bryostatin 1 (henceforth bryostatin is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

Science.gov (United States)

Arias, C A; Martín-Martinez, M; Blundell, T L; Arthur, M; Courvalin, P; Reynolds, P E

1999-03-01

Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney

International Nuclear Information System (INIS)

Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A.

1988-01-01

A partially purified, membrane-bound Na + -K + -ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H 2 O 2 for either 15 or 30 min at 37 degree C. The activity of ouabain-sensitive Na + -K + -ATPase was reduced proportionally to the concentration of H 2 O 2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na + -K + -ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na + K + -ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degree C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na + -K + -ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney

NMR structure of temporin-1 ta in lipopolysaccharide micelles: mechanistic insight into inactivation by outer membrane.

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Rathi Saravanan

Full Text Available BACKGROUND: Antimicrobial peptides (AMPs play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS. The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane. PRINCIPAL FINDINGS: Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles. SIGNIFICANCE: We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a

NMR structural studies of peptides and proteins in membranes

Energy Technology Data Exchange (ETDEWEB)

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

1994-12-31

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

Sandwich-structured hollow fiber membranes for osmotic power generation

KAUST Repository

Fu, Feng Jiang; Zhang, Sui; Chung, Neal Tai-Shung

2015-01-01

In this work, a novel sandwich-structured hollow fiber membrane has been developed via a specially designed spinneret and optimized spinning conditions. With this specially designed spinneret, the outer layer, which is the most crucial part of the sandwich-structured membrane, is maintained the same as the traditional dual-layer membrane. The inner substrate layer is separated into two layers: (1) an ultra-thin middle layer comprising a high molecular weight polyvinylpyrrolidone (PVP) additive to enhance integration with the outer polybenzimidazole (PBI) selective layer, and (2) an inner-layer to provide strong mechanical strength for the membrane. Experimental results show that a high water permeability and good mechanical strength could be achieved without the expensive post treatment process to remove PVP which was necessary for the dual-layer pressure retarded osmosis (PRO) membranes. By optimizing the composition, the membrane shows a maximum power density of 6.23W/m2 at a hydraulic pressure of 22.0bar when 1M NaCl and 10mM NaCl are used as the draw and feed solutions, respectively. To our best knowledge, this is the best phase inversion hollow fiber membrane with an outer selective PBI layer for osmotic power generation. In addition, this is the first work that shows how to fabricate sandwich-structured hollow fiber membranes for various applications. © 2015 Elsevier B.V.

Sandwich-structured hollow fiber membranes for osmotic power generation

KAUST Repository

Fu, Feng Jiang

2015-11-01

In this work, a novel sandwich-structured hollow fiber membrane has been developed via a specially designed spinneret and optimized spinning conditions. With this specially designed spinneret, the outer layer, which is the most crucial part of the sandwich-structured membrane, is maintained the same as the traditional dual-layer membrane. The inner substrate layer is separated into two layers: (1) an ultra-thin middle layer comprising a high molecular weight polyvinylpyrrolidone (PVP) additive to enhance integration with the outer polybenzimidazole (PBI) selective layer, and (2) an inner-layer to provide strong mechanical strength for the membrane. Experimental results show that a high water permeability and good mechanical strength could be achieved without the expensive post treatment process to remove PVP which was necessary for the dual-layer pressure retarded osmosis (PRO) membranes. By optimizing the composition, the membrane shows a maximum power density of 6.23W/m2 at a hydraulic pressure of 22.0bar when 1M NaCl and 10mM NaCl are used as the draw and feed solutions, respectively. To our best knowledge, this is the best phase inversion hollow fiber membrane with an outer selective PBI layer for osmotic power generation. In addition, this is the first work that shows how to fabricate sandwich-structured hollow fiber membranes for various applications. © 2015 Elsevier B.V.

Peripheral Protein Unfolding Drives Membrane Bending.

Science.gov (United States)

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Self-assembling peptides form nanodiscs that stabilize membrane proteins

DEFF Research Database (Denmark)

Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

2014-01-01

-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

Microwave-Driven Multifunctional Capability of Membrane Structures

Science.gov (United States)

Choi, Sang H.; Chu, Sang-Hyong; Song, Kyo D.; King, Glen C.

2002-01-01

A large, ultra lightweight space structure, such as solar sails and Gossamer spacecrafts, requires a distributed power source to alleviate wire networks, unlike the localized on-board power infrastructures typically found in most small spacecrafts. The concept of microwave-driven multifunctional capability for membrane structures is envisioned as the best option to alleviate the complexity associated with hard-wired control circuitry and on-board power infrastructures. A rectenna array based on a patch configuration for high voltage output was developed to drive membrane actuators, sensors, probes, or other devices. Networked patch rectenna array receives and converts microwave power into a DC power for an array of smart actuators. To use microwave power effectively, the concept of a power allocation and distribution (PAD) circuit is adopted for networking a rectenna/actuator patch array. The use of patch rectennas adds a significant amount of rigidity to membrane flexibility and they are relatively heavy. A dipole rectenna array (DRA) appears to be ideal for thin-film membrane structures, since DRA is flexible and light. Preliminary design and fabrication of PAD circuitry that consists of a few nodal elements were made for laboratory testing. The networked actuators were tested to correlate the network coupling effect, power allocation and distribution, and response time.

Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

Directory of Open Access Journals (Sweden)

Roberts Paul C

2009-04-01

Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

Antibacterial Membrane with a Bone-Like Structure for Guided Bone Regeneration

Directory of Open Access Journals (Sweden)

YuYuan Zhang

2015-01-01

Full Text Available An antibacterial membrane with a bone-like structure was developed for guided bone regeneration (GBR by mineralising acellular bovine pericardium (ABP and loading it with the antibiotic minocycline. The bovine pericardium (BP membrane was processed using physical and chemical methods to remove the cellular components and obtain ABP membranes. Then, the ABP membranes were biomimetically mineralised using a calcium phosphate-loaded agarose hydrogel system aided by electrophoresis. Minocycline was adsorbed to the mineralised ABP membrane, and the release profile in vitro was studied. The membranes were characterised through scanning electron microscopy, diffuse reflectance-Fourier transform infrared spectroscopy, and X-ray diffraction. Results showed that the ABP membrane had an asymmetric structure with a layer of densely arranged and irregularly aligned collagen fibrils. Collagen fibrils were calcified with the formation of intrafibrillar and interfibrillar hydroxyapatites similar to the bone structure. Minocycline was incorporated into the mineralised collagen membrane and could be released in vitro. This process endowed the membrane with an antibacterial property. This novel composite membrane offers promising applications in bioactive GBR.

A structural and kinetic link between membrane association and amyloid fibril formation of α-Synuclein

OpenAIRE

Heise, Henrike; Etzkorn, Manuel; Hoyer, Wolfgang; Buell, Alexander; Strodel, Birgit; Willbold, Dieter; Shaykhalishahi, Hamed; Poojari, Chetan; Uluca, Boran; Wördehoff, Michael; Viennet, Thibault

2017-01-01

The protein α-Synuclein (αS) is linked to Parkinson's disease through its abnormal aggregation, which is thought to involve an interplay between cytosolic and membrane-bound forms of αS. Therefore, better insights into the molecular determinants of membrane association and their implications for protein aggregation may help deciphering the pathogenesis of Parkinson's disease. Following previous studies using micelles and vesicles, we present a comprehensive study of αS interaction with phosph...

Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

Science.gov (United States)

Sahu, Indra D; Zhang, Rongfu; Dunagan, Megan M; Craig, Andrew F; Lorigan, Gary A

2017-06-01

EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (T m ) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the T m titration experiment indicated an increase in T m values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant

Carbon nanotube embedded PVDF membranes: Effect of solvent composition on the structural morphology for membrane distillation

Science.gov (United States)

Mapunda, Edgar C.; Mamba, Bhekie B.; Msagati, Titus A. M.

2017-08-01

Rapid population increase, growth in industrial and agricultural sectors and global climate change have added significant pressure on conventional freshwater resources. Tapping freshwater from non-conventional water sources such as desalination and wastewater recycling is considered as sustainable alternative to the fundamental challenges of water scarcity. However, affordable and sustainable technologies need to be applied for the communities to benefit from the treatment of non-conventional water source. Membrane distillation is a potential desalination technology which can be used sustainably for this purpose. In this work multi-walled carbon nanotube embedded polyvinylidene fluoride membranes for application in membrane distillation desalination were prepared via non-solvent induced phase separation method. The casting solution was prepared using mixed solvents (N, N-dimethylacetamide and triethyl phosphate) at varying ratios to study the effect of solvent composition on membrane morphological structures. Membrane morphological features were studied using a number of techniques including scanning electron microscope, atomic force microscope, SAXSpace tensile strength analysis, membrane thickness, porosity and contact angle measurements. It was revealed that membrane hydrophobicity, thickness, tensile strength and surface roughness were increasing as the composition of N, N-dimethylacetamide in the solvent was increasing with maximum values obtained between 40 and 60% N, N-dimethylacetamide. Internal morphological structures were changing from cellular structures to short finger-like and sponge-like pores and finally to large macro void type of pores when the amount of N, N-dimethylacetamide in the solvent was changed from low to high respectively. Multi-walled carbon nanotube embedded polyvinylidene fluoride membranes of desired morphological structures and physical properties can be synthesized by regulating the composition of solvents used to prepare the

Calculation of separation selectivity of aqueous electrolytic solutions with reverse osmosis membranes

International Nuclear Information System (INIS)

Ognevskij, A.V.; Fomichev, S.V.; Khvostov, V.F.; Kochergin, N.V.; AN SSSR, Moscow

1988-01-01

Viscosity and dielectric permittivity of a bound water layer in micropores of cellulose acetate membranes used for electrolyte ion separation by reverse osmosis method are calculated using the water cluster model and the proposed structural temperature parameter. Based on the model representations presented an algorithmof reverse osmosis membrane selectivity calculation in diluted aqueous solutions ofelectrolytes containing Cs + , Sr 2+ , I - and other ions is constructed

Structure and Dynamic Properties of Membrane Proteins using NMR

DEFF Research Database (Denmark)

Rösner, Heike; Kragelund, Birthe

2012-01-01

conformational changes. Their structural and functional decoding is challenging and has imposed demanding experimental development. Solution nuclear magnetic resonance (NMR) spectroscopy is one of the techniques providing the capacity to make a significant difference in the deciphering of the membrane protein...... structure-function paradigm. The method has evolved dramatically during the last decade resulting in a plethora of new experiments leading to a significant increase in the scientific repertoire for studying membrane proteins. Besides solving the three-dimensional structures using state-of-the-art approaches......-populated states, this review seeks to introduce the vast possibilities solution NMR can offer to the study of membrane protein structure-function analyses with special focus on applicability. © 2012 American Physiological Society. Compr Physiol 2:1491-1539, 2012....

Robust mixed conducting membrane structure

DEFF Research Database (Denmark)

2010-01-01

circuited. The present invention further provides a method of producing the above membrane structure, comprising the steps of : providing a ionically conducting layer; applying at least one layer of electronically conducting material on each side of said ionically conducting layer; sintering the multilayer...

Molecular Structure of Membrane Tethers

NARCIS (Netherlands)

Baoukina, Svetlana; Marrink, Siewert J.; Tieleman, D. Peter

2012-01-01

Membrane tethers are nanotubes formed by a lipid bilayer. They play important functional roles in cell biology and provide an experimental window on lipid properties. Tethers have been studied extensively in experiments and described by theoretical models, but their molecular structure remains

Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

International Nuclear Information System (INIS)

Zambetti, G.; Stein, J.; Stein, G.

1987-01-01

The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

Energy Technology Data Exchange (ETDEWEB)

Ruskamo, Salla [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Yadav, Ravi P. [Banaras Hindu University, Varanasi (India); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Sharma, Satyan; Lehtimäki, Mari [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Laulumaa, Saara [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Aggarwal, Shweta; Simons, Mikael [Max Planck Institute for Experimental Medicine, Göttingen (Germany); Bürck, Jochen; Ulrich, Anne S. [Karlsruhe Institute for Technology (KIT), Karlsruhe (Germany); Juffer, André H. [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Kursula, Inari [University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Kursula, Petri, E-mail: petri.kursula@oulu.fi [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); University of Hamburg, Hamburg (Germany)

2014-01-01

The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.

Structural studies of Ca2+-ATPase ligand and regulatory complexes

DEFF Research Database (Denmark)

Drachmann, Nikolaj Düring

2015-01-01

, the surrounding membrane itself has a huge influence on SERCA structure and function. Changes in the membrane thickness can alter the activity of the ATPase significantly, and even cause changes in the stoichiometry of ion transport. Structural studies on SERCA in the presence of four different phosphatidyl...... choline lipids with different aliphatic chain length and saturation show three specific lipid binding sites. The four different lipids analysed bind to the same binding sites with varying degrees of disorder. The study contributes to understanding the complex interplay between the surrounding membrane...... to explore the possibilities for an efficient screening of ligand-bound SERCA structures, serial femtosecond crystallography experiments of microcrystals of SERCA1a in the Ca2+ bound state and in a vanadate stabilised E2 state was conducted. A structure obtained at 2.8 Å maximum resolution of the proof...

Aggressive re-warming at 38.5 degrees C following deep hypothermia at 21 degrees C increases neutrophil membrane bound elastase activity and pro-inflammatory factor release

NARCIS (Netherlands)

Tang, Min; Zhao, Xiao-gang; He, Yi; Gu, Yan; Mei, Ju

2016-01-01

Background: Cardiopulmonary bypass (CPB) is often performed under hypothermic condition. The effects of hypothermia and re-warming on neutrophil activity are unclear. This study aimed to compare the effects of different hypothermia and re-warming regimens on neutrophil membrane bound elastase (MBE)

Free and membrane-bound ribosomes and polysomes in hippocampal neurons during a learning experiment.

Science.gov (United States)

Wenzel, J; David, H; Pohle, W; Marx, I; Matthies, H

1975-01-24

The ribosomes of the CA1 and CA3 pyramidal cells of hipocampus were investigated by morphometric methods after the acquisition of a shock-motivated brightness discrimination in rats. A significant increase in the total number of ribosomes was observed in CA1 cells of trained animals and in CA3 cells of both active controls and trained rats. A significant increase in membrane-bound ribosomes was obtained in CA1 and CA3 cells after training only. The results confirm the suggestion of an increased protein synthesis in hippocampal neurons during and after the acquisition of a brightness discrimination, as we have concluded from out previous investigations on the incorporation of labeled amino acids under identical experimental conditions. The results lead to the assumption that the protein synthesis in some neuronal cells may probably differ not only quantitatively, but also qualitatively in trained and untrained animals.

Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

Science.gov (United States)

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

Development of topologically structured membranes of aluminum oxide

Science.gov (United States)

Bankova, A.; Videkov, V.; Tzaneva, B.

2014-05-01

In recent years, nanomembranes have become one of the most widely used construction material for ultrasensitive and ultrathin applications in micro-electromechanical systems (MEMS) and other sensor structures due to their remarkable mechanical properties. Among these, the mechanical stability is of particular importance. We present an approach to the analysis of the stability of nanostructured anodic aluminum oxide free membranes subjected to mechanical bending. The membranes tested were with a thickness of 500 nm to 15 urn in various topological shapes; we describe the technological schemes of their preparation. Bends were applied to membranes prepared by using a selective process of etching and anodizing. The results of the preparation of the membranes are discussed, together with the influence of the angle of deflection, and the number of bendings. The results obtained can be used in designing MEMS structures and sensors which use nanostructured anodic aluminum oxide.

Development of topologically structured membranes of aluminum oxide

International Nuclear Information System (INIS)

Bankova, A; Videkov, V; Tzaneva, B

2014-01-01

In recent years, nanomembranes have become one of the most widely used construction material for ultrasensitive and ultrathin applications in micro-electromechanical systems (MEMS) and other sensor structures due to their remarkable mechanical properties. Among these, the mechanical stability is of particular importance. We present an approach to the analysis of the stability of nanostructured anodic aluminum oxide free membranes subjected to mechanical bending. The membranes tested were with a thickness of 500 nm to 15 urn in various topological shapes; we describe the technological schemes of their preparation. Bends were applied to membranes prepared by using a selective process of etching and anodizing. The results of the preparation of the membranes are discussed, together with the influence of the angle of deflection, and the number of bendings. The results obtained can be used in designing MEMS structures and sensors which use nanostructured anodic aluminum oxide.

Membrane transport mechanism 3D structure and beyond

CERN Document Server

Ziegler, Christine

2014-01-01

This book provides a molecular view of membrane transport by means of numerous biochemical and biophysical techniques. The rapidly growing number of atomic structures of transporters in different conformations and the constant progress in bioinformatics have recently added deeper insights.   The unifying mechanism of energized solute transport across membranes is assumed to consist of the conformational cycling of a carrier protein to provide access to substrate binding sites from either side of a cellular membrane. Due to the central role of active membrane transport there is considerable interest in deciphering the principles of one of the most fundamental processes in nature: the alternating access mechanism.   This book brings together particularly significant structure-function studies on a variety of carrier systems from different transporter families: Glutamate symporters, LeuT-like fold transporters, MFS transporters and SMR (RND) exporters, as well as ABC-type importers.   The selected examples im...

Metabolism of fatty acids in rat brain in microsomal membranes

International Nuclear Information System (INIS)

Aeberhard, E.E.; Gan-Elepano, M.; Mead, J.F.

1980-01-01

Using a technique in which substrate fatty acids are incorporated into microsomal membranes followd by comparison of their rates of desaturation or elongation with those of exogenous added fatty acids it has been found that the desaturation rate is more rapid for the membrane-bound substrate than for the added fatty acid. Moreover, the product of the membrane-bound substrate is incorporated into membrane phospholipid whereas the product of the exogenous substrate is found in di- and triacyl glycerols and in free fatty acids as well. These and other findings point to a normal sequence of reaction of membrane liqids with membrane-bound substrates involving transfer of fatty acid from phospholipid to the coupled enzyme systems without ready equilibration with the free fatty acid pool

Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

Directory of Open Access Journals (Sweden)

Nimgampalle Mallikarjuna

2016-12-01

Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20% in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration.

Scattering by bound nucleons

International Nuclear Information System (INIS)

Tezuka, Hirokazu.

1984-10-01

Scattering of a particle by bound nucleons is discussed. Effects of nucleons that are bound in a nucleus are taken as a structure function. The way how to calculate the structure function is given. (author)

Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II recepter HLA-DR1

Energy Technology Data Exchange (ETDEWEB)

Mullen, M.; Haan, K.M.; Longnecker, R.; Jardetzky, T.

2010-03-08

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

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

Science.gov (United States)

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

2018-05-01

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

Overcoming bottlenecks in the membrane protein structural biology pipeline.

Science.gov (United States)

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

2016-06-15

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

Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.

Science.gov (United States)

Xia, Yan; Fischer, Axel W; Teixeira, Pedro; Weiner, Brian; Meiler, Jens

2018-04-03

While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available. Copyright © 2018 Elsevier Ltd. All rights reserved.

Changes in plasma membrane structure upon irradiation on thymocytes

International Nuclear Information System (INIS)

Dreval', V.I.

1993-01-01

Thymocytes were irradiated with doses of 4 to 10 4 Gy. The binding of 1-anilinonaphtalene-8-sulphonate and Ca 2+ to plasma membranes; viscosity and lipid peroxidation; Stern-Folmer constant; and the number of Sh-groups of membrane proteins were determined. The structural changes in plasma membranes after irradiation of thymocytes were found to be cooperative

Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes.

Science.gov (United States)

Pankov, R; Markovska, T; Antonov, P; Ivanova, L; Momchilova, A

2006-09-01

Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers.

Correct use of Membrane Elements in Structural Analysis

Directory of Open Access Journals (Sweden)

Rothman Timothy

2016-01-01

Full Text Available Structural analysis of consumer electronic devices such as phones and tablets involves Finite Element Analysis (FEA. Dynamic loading conditions such as device dropping and bending dictate accurate FEA models to reduce design risk in many areas. The solid elements typically used in structural analysis do not have integration points on the surface. The outer surface is of most interest because that is where the cracks start. Analysts employ a post processing trick through using membranes to bring accurate stress/strain results to the surface. This paper explains numerical issues with implementation of membranes and recommends a methodology for accurate structural analysis.

FATE OF REVERSE OSMOSIS (RO) MEMBRANES DURING OXIDATION BY DISINFECTANTS USED IN WATER TREATMENT: IMPACT ON MEMBRANE STRUCTURE AND PERFORMANCES

KAUST Repository

Maugin, Thomas

2013-12-01

Providing pretreatment prior RO filtration is essential to avoid biofouling and subsequent loss of membrane performances. Chlorine is known to degrade polymeric membrane, improving or reducing membrane efficiency depending on oxidation conditions. This study aimed to assess the impact of alternative disinfectant, NH2Cl, as well as secondary oxidants formed during chloramination of seawater, e.g. HOBr, HOI, or used in water treatment e.g. ClO2, O3, on membrane structure and performances. Permeability, total and specific rejection (Cl-, SO4 2-, Br-, Boron), FTIR profile, elemental composition were analyzed. Results showed that each oxidant seems to react differently with the membrane. HOCl, HOBr, ClO2 and O3 improved membrane permeability but decreased rejection in different extent. In comparison, chloramines resulted in identical trends but oxidized membrane very slowly. On the contrary, iodine improved membrane rejection e.g. boron, but decreased permeability. Reaction conducted with chlorine, bromine, iodine and chloramines resulted in the incorporation of halogen in the membrane structure. All oxidant except iodine were able to break amide bonds of the membrane structure in our condition. In addition, chloramine seemed to react with membrane differently, involving a potential addition of nitrogen. Chloramination of seawater amplified membrane performances evolutions due to generation of bromochloramine. Moreover, chloramines reacted both with NOM and membrane during oxidation in natural seawater, leading to additional rejection drop.

Peptide-membrane Interactions by Spin-labeling EPR

Science.gov (United States)

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

DNA nanotubes for NMR structure determination of membrane proteins.

Science.gov (United States)

Bellot, Gaëtan; McClintock, Mark A; Chou, James J; Shih, William M

2013-04-01

Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.

Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

Science.gov (United States)

Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

2017-10-01

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

Towards Co-evolution of Membranes and Metabolism

Science.gov (United States)

Wei, Chenyu; Wilson, Michael A.; Pohorille, Andrew

2014-01-01

Conceptually, the most robust way to explain how primitive cell-like structures acquired and increased their capabilities is on the basis of Darwinian evolution. A population of protocells containing material that produced more environmentally fit progeny would increase in time at the expense of other protocells. In this scenario, protocellular boundaries were inextricably connected to the metabolism they encapsulated: to be inheritable, early metabolism must have led to an increased rate of growth and division of vesicles and, similarly, transport through vesicle boundaries must have supported the evolution of metabolism. Everything that could not be delivered from the environment had to be produced and retained inside protocells. Despite their importance to the understanding of the origin of life, only a few cases of coupling between metabolism and membrane-related processes have been identified so far. For example, reactions inside fatty-acid vesicles have been linked to their competitive growth and division, and mechanisms by which membrane permeability might have coupled to information polymers have been proposed and explained. Most recently, it has been shown that a dipeptide inside fatty-acid vesicles catalyzes the formation of another dipeptide that binds to vesicle walls and, by doing so, promotes their growth at the expense of other vesicles, thus demonstrating evolutionary advantage of small, membrane-bound peptides. It has been shown that the appearance of phospholipids imparted selective advantage to protocells bound by phospholipid-containing membranes, eventually driving fatty-acid vesicles to extinction. Phospholipid membranes, however, are nearly impermeable to charged species. Yet, the ability to transport ions across membranes was vital for regulating cellular volume, pH homeostasis, generating energy and sensing the environment. To account for this, evolutionary scenarios for the emergence of simple ion channels, protein structures surrounding

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

Science.gov (United States)

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

2017-09-01

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

Structural Study and Modification of Support Layer for Forward Osmosis Membranes

KAUST Repository

Shi, Meixia

2016-01-01

polymerization. Among the different substrates we include standard asymmetric porous membranes prepared from homopolymers, such as polysulfone. Additionally block copolymer membrane and Anodisc alumina membrane are chosen based on their exceptional structures

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

Science.gov (United States)

Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

2012-01-01

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

MEMS-Based Fuel Reformer with Suspended Membrane Structure

Science.gov (United States)

Chang, Kuei-Sung; Tanaka, Shuji; Esashi, Masayoshi

We report a MEMS-based fuel reformer for supplying hydrogen to micro-fuel cells for portable applications. A combustor and a reforming chamber are fabricated at either side of a suspended membrane structure. This design is used to improve the overall thermal efficiency, which is a critical issue to realize a micro-fuel reformer. The suspended membrane structure design provided good thermal isolation. The micro-heaters consumed 0.97W to maintain the reaction zone of the MEMS-based fuel reformer at 200°C, but further power saving is necessary by improving design and fabrication. The conversion rate of methanol to hydrogen was about 19% at 180°C by using evaporated copper as a reforming catalyst. The catalytic combustion of hydrogen started without any assistance of micro-heaters. By feeding the fuel mixture of an equivalence ratio of 0.35, the temperature of the suspended membrane structure was maintained stable at 100°C with a combustion efficiency of 30%. In future works, we will test a micro-fuel reformer by using a micro-combustor to supply heat.

Membrane-based ethylene/ethane separation: The upper bound and beyond

KAUST Repository

Rungta, Meha; Zhang, Chen; Koros, William J.; Xu, Liren

2013-01-01

Ethylene/ethane separation via cryogenic distillation is extremely energy-intensive, and membrane separation may provide an attractive alternative. In this paper, ethylene/ethane separation performance using polymeric membranes is summarized

Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

Science.gov (United States)

Sun, Xiaoou; Wiesner, Burkhard; Lorenz, Dorothea; Papsdorf, Gisela; Pankow, Kristin; Wang, Po; Dietrich, Nils; Siems, Wolf-Eberhard; Maul, Björn

2008-12-01

Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

Role of charged lipids in membrane structures — Insight given by simulations

DEFF Research Database (Denmark)

Pöyry, Sanja; Vattulainen, Ilpo

2016-01-01

Lipids and proteins are the main components of cell membranes. It is becoming increasingly clear that lipids, in addition to providing an environment for proteins to work in, are in many cases also able to modulate the structure and function of those proteins. Particularly charged lipids...... to fruitful directions. In this paper, we review studies that have utilized molecular dynamics simulations to unravel the roles of charged lipids in membrane structures. We focus on lipids as active constituents of the membranes, affecting both general membrane properties as well as non-lipid membrane...

Characterization of a structural intermediate of flavivirus membrane fusion.

Directory of Open Access Journals (Sweden)

Karin Stiasny

2007-02-01

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

Hydroxycinnamic acid bound arabinoxylans from millet brans-structural features and antioxidant activity.

Science.gov (United States)

Bijalwan, Vandana; Ali, Usman; Kesarwani, Atul Kumar; Yadav, Kamalendra; Mazumder, Koushik

2016-07-01

Hydroxycinnamic acid bound arabinoxylans (HCA-AXs) were extracted from brans of five Indian millet varieties and response surface methodology was used to optimize the extraction conditions. The optimal condition to obtain highest yield of millet HCA-AXs was determined as follows: time 61min, temperature 66°C, ratio of solvent to sample 12ml/g. Linkage analysis indicated that hydroxycinnamic acid bound arabinoxylan from kodo millet (KM-HCA-AX) contained comparatively low branched arabinoxylan consisting of 14.6% mono-substituted, 1.2% di-substituted and 41.2% un-substituted Xylp residues. The HPLC analysis of millet HCA-AXs showed significant variation in the content of three major bound hydroxycinnamic acids (caffeic, p-coumaric and ferulic acid). The antioxidant activity of millet HCA-AXs were evaluated using three in vitro assay methods (DPPH, FRAP and β-carotene linoleate emulsion assays) which suggested both phenolic acid composition and structural characteristics of arabinoxylans could be correlated to their antioxidant potential, the detailed structural analysis revealed that low substituted KM-HCA-AX exhibited relatively higher antioxidant activity compared to other medium and highly substituted HCA-AXs from finger (FM), proso (PM), barnyard (BM) and foxtail (FOXM) millet. Copyright © 2016. Published by Elsevier B.V.

Membrane interaction and functional plasticity of inositol polyphosphate 5-phosphatases.

Science.gov (United States)

Braun, Werner; Schein, Catherine H

2014-05-06

In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. Copyright © 2014 Elsevier Ltd. All rights reserved.

Single-particle electron microscopy in the study of membrane protein structure.

Science.gov (United States)

De Zorzi, Rita; Mi, Wei; Liao, Maofu; Walz, Thomas

2016-02-01

Single-particle electron microscopy (EM) provides the great advantage that protein structure can be studied without the need to grow crystals. However, due to technical limitations, this approach played only a minor role in the study of membrane protein structure. This situation has recently changed dramatically with the introduction of direct electron detection device cameras, which allow images of unprecedented quality to be recorded, also making software algorithms, such as three-dimensional classification and structure refinement, much more powerful. The enhanced potential of single-particle EM was impressively demonstrated by delivering the first long-sought atomic model of a member of the biomedically important transient receptor potential channel family. Structures of several more membrane proteins followed in short order. This review recounts the history of single-particle EM in the study of membrane proteins, describes the technical advances that now allow this approach to generate atomic models of membrane proteins and provides a brief overview of some of the membrane protein structures that have been studied by single-particle EM to date. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Resolving the Spatial Structures of Bound Hole States in Black Phosphorus.

Science.gov (United States)

Qiu, Zhizhan; Fang, Hanyan; Carvalho, Alexandra; Rodin, A S; Liu, Yanpeng; Tan, Sherman J R; Telychko, Mykola; Lv, Pin; Su, Jie; Wang, Yewu; Castro Neto, A H; Lu, Jiong

2017-11-08

Understanding the local electronic properties of individual defects and dopants in black phosphorus (BP) is of great importance for both fundamental research and technological applications. Here, we employ low-temperature scanning tunnelling microscope (LT-STM) to probe the local electronic structures of single acceptors in BP. We demonstrate that the charge state of individual acceptors can be reversibly switched by controlling the tip-induced band bending. In addition, acceptor-related resonance features in the tunnelling spectra can be attributed to the formation of Rydberg-like bound hole states. The spatial mapping of the quantum bound states shows two distinct shapes evolving from an extended ellipse shape for the 1s ground state to a dumbbell shape for the 2p x excited state. The wave functions of bound hole states can be well-described using the hydrogen-like model with anisotropic effective mass, corroborated by our theoretical calculations. Our findings not only provide new insight into the many-body interactions around single dopants in this anisotropic two-dimensional material but also pave the way to the design of novel quantum devices.

Shell closures, loosely bound structures, and halos in exotic nuclei

International Nuclear Information System (INIS)

Saxena, G.; Singh, D.

2013-01-01

Inspired by the recent experiments indicating doubly magic nuclei that lie near the drip-line and encouraged by the success of our relativistic mean-field (RMF) plus state-dependent BCS approach to the description of the ground-state properties of drip-line nuclei, we develop this approach further, across the entire periodic table, to explore magic nuclei, loosely bound structures, and halo formation in exotic nuclei. In our RMF+BCS approach, the single-particle continuum corresponding to the RMF is replaced by a set of discrete positive-energy states for the calculations of pairing energy. Detailed analysis of the single-particle spectrum, pairing energies, and densities of the nuclei predict the unusual proton shell closures at proton numbers Z = 6, 14, 16, 34, and unusual neutron shell closures at neutron numbers N = 6, 14, 16, 34, 40, 70, 112. Further, in several nuclei like the neutron-rich isotopes of Ca, Zr, Mo, etc., the gradual filling of lowlying single-particle resonant state together with weakly bound single-particle states lying close to the continuum threshold helps accommodate more neutrons but with an extremely small increase in the binding energy. This gives rise to the occurrence of loosely bound systems of neutron-rich nuclei with a large neutron-to-proton ratio. In general, the halo-like formation, irrespective of the existence of any resonant state, is seen to be due to the large spatial extension of the wave functions for the weakly bound single-particle states with low orbital angular momentum having very small or no centrifugal barriers.

Shell closures, loosely bound structures, and halos in exotic nuclei

Energy Technology Data Exchange (ETDEWEB)

Saxena, G., E-mail: gauravphy@gmail.com [Govt. Women Engineering College, Department of Physics (India); Singh, D. [University of Rajasthan, Department of Physics (India)

2013-04-15

Inspired by the recent experiments indicating doubly magic nuclei that lie near the drip-line and encouraged by the success of our relativistic mean-field (RMF) plus state-dependent BCS approach to the description of the ground-state properties of drip-line nuclei, we develop this approach further, across the entire periodic table, to explore magic nuclei, loosely bound structures, and halo formation in exotic nuclei. In our RMF+BCS approach, the single-particle continuum corresponding to the RMF is replaced by a set of discrete positive-energy states for the calculations of pairing energy. Detailed analysis of the single-particle spectrum, pairing energies, and densities of the nuclei predict the unusual proton shell closures at proton numbers Z = 6, 14, 16, 34, and unusual neutron shell closures at neutron numbers N = 6, 14, 16, 34, 40, 70, 112. Further, in several nuclei like the neutron-rich isotopes of Ca, Zr, Mo, etc., the gradual filling of lowlying single-particle resonant state together with weakly bound single-particle states lying close to the continuum threshold helps accommodate more neutrons but with an extremely small increase in the binding energy. This gives rise to the occurrence of loosely bound systems of neutron-rich nuclei with a large neutron-to-proton ratio. In general, the halo-like formation, irrespective of the existence of any resonant state, is seen to be due to the large spatial extension of the wave functions for the weakly bound single-particle states with low orbital angular momentum having very small or no centrifugal barriers.

High throughput platforms for structural genomics of integral membrane proteins.

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Mancia, Filippo; Love, James

2011-08-01

Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

2016-01-01

Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic potential within phospholipid membranes imply an enormous electric field of 10 8 –10 9 V m −1 , which is likely to have great significance in controlling the conformation of translocating membrane proteins and in the transfer of ions and molecules across the membrane. We have calculated the membrane potential for DMPG bilayers and found ∼1 V (∼2 ⋅ 10 8 V m −1 ) when in the fluid phase with a monovalent counter-ion and ∼1.4 V (∼2.8 ⋅ 10 8 V m −1 ) when in the gel phase with a divalent counter-ion. The number of water molecules for a fully hydrated DMPG membrane has been estimated to be 9.7 molecules per lipid in the gel phase and 17.5 molecules in the fluid phase, considerably smaller than inferred experimentally for 1,2-dimyristoyl-sn-glycero-3

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-04-14

Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic potential within phospholipid membranes imply an enormous electric field of 10{sup 8}–10{sup 9} V m{sup −1}, which is likely to have great significance in controlling the conformation of translocating membrane proteins and in the transfer of ions and molecules across the membrane. We have calculated the membrane potential for DMPG bilayers and found ∼1 V (∼2 ⋅ 10{sup 8} V m{sup −1}) when in the fluid phase with a monovalent counter-ion and ∼1.4 V (∼2.8 ⋅ 10{sup 8} V m{sup −1}) when in the gel phase with a divalent counter-ion. The number of water molecules for a fully hydrated DMPG membrane has been estimated to be 9.7 molecules per lipid in the gel phase and 17.5 molecules in the fluid phase, considerably smaller than inferred experimentally for 1

The structure and function of cell membranes studied by atomic force microscopy.

Science.gov (United States)

Shi, Yan; Cai, Mingjun; Zhou, Lulu; Wang, Hongda

2018-01-01

The cell membrane, involved in almost all communications of cells and surrounding matrix, is one of the most complicated components of cells. Lack of suitable methods for the detection of cell membranes in vivo has sparked debates on the biochemical composition and structure of cell membranes over half a century. The development of single molecule techniques, such as AFM, SMFS, and TREC, provides a versatile platform for imaging and manipulating cell membranes in biological relevant environments. Here, we discuss the latest developments in AFM and the progress made in cell membrane research. In particular, we highlight novel structure models and dynamic processes, including the mechanical properties of the cell membranes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.

Directory of Open Access Journals (Sweden)

Kap Lim

Full Text Available The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP, was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170, which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.

Depression of membrane-bound Na sup + -K sup + -ATPase activity induced by free radicals and by ischemia of kidney

Energy Technology Data Exchange (ETDEWEB)

Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A. (Univ. of Ottawa, Ontario (Canada))

1988-02-01

A partially purified, membrane-bound Na{sup +}-K{sup +}-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H{sub 2}O{sub 2} for either 15 or 30 min at 37{degree}C. The activity of ouabain-sensitive Na{sup +}-K{sup +}-ATPase was reduced proportionally to the concentration of H{sub 2}O{sub 2} and the duration of incubation. There were decreases in SH contents and turnover rates of the Na{sup +}-K{sup +}-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na{sup +}K{sup +}-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4{degree}C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na{sup +}-K{sup +}-ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney.

PDMS/PVDF hybrid electrospun membrane with superhydrophobic property and drop impact dynamics for dyeing wastewater treatment using membrane distillation

KAUST Repository

An, Alicia Kyoungjin

2016-10-21

Fouling in membrane distillation (MD) results in an increase in operation costs and deterioration in a water quality. In this work, a poly(vinylidene fluoride-co-hexafluoropropene) (PVDF-HFP) electrospun (E-PH) membrane was fabricated by hybridizing polydimethylsiloxane (PDMS) polymeric microspheres with superhydrophobicity onto the E-PH membrane via electrospinning. The resulting hybrid PDMS with E-PH (E-PDMS) membrane showed a significant enhancement in surface hydrophobicity (contact angle, CA = 155.4°) and roughness (Ra = 1,285mm). The zeta potential of E-PDMS membrane surface showed a higher negative value than that of a commercial PVDF (C-PVDF) membrane. These properties of E-PDMS membrane provided an antifouling in treating of differently-charged dyes and generated a flake-like dye–dye (loosely bound foulant) structure on the membrane surface rather than in the membrane pores. This also led to a high productivity of E-PDMS membrane (34 Lm-2h-1, 50% higher than that of C-PVDF membrane) without fouling or wetting. In addition, complete color removal and pure water production were achieved during a long-term operation. An application of intermittent water flushing (WF) in direct contact MD (DCMD) operation led to a 99% CA recovery of E-PDMS membrane indicating its sustainability. Therefore, the E-PDMS membrane is a promising candidate for MD application in dyeing wastewater treatment.

Current strategies for protein production and purification enabling membrane protein structural biology.

Science.gov (United States)

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Plasma-polymerized alkaline anion-exchange membrane: Synthesis and structure characterization

International Nuclear Information System (INIS)

Hu Jue; Meng Yuedong; Zhang Chengxu; Fang Shidong

2011-01-01

After-glow discharge plasma polymerization was developed for alkaline anion-exchange membranes synthesis using vinylbenzyl chloride as monomer. X-ray photoelectron spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were used to characterize the chemical structure properties of plasma-polymerized membranes. Ion-exchange capacities of quaternized poly(vinylbenzyl chloride) (QPVBC) membranes were measured to evaluate their capability of hydroxyl ion transport. A mechanism of plasma polymerization using VBC as monomer that accounts for the competitive effects of free radicals polymerization and plasma ablation in the plasma polymerization process was proposed. Our results indicate that plasma discharge power influences the contents of functional groups and the structure of the plasma polymer membranes, which attribute to the coactions of polymerization and ablation. The properties of uniform morphology, good adhesion to the substrate, high thermal stability and satisfying anion conduction level suggest the potential application of QPVBC membrane deposited at discharge power of 20 W in alkaline direct methanol fuel cells.

Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking.

Science.gov (United States)

Ducassou, Lionel; Dhers, Laura; Jonasson, Gabriella; Pietrancosta, Nicolas; Boucher, Jean-Luc; Mansuy, Daniel; André, François

2017-09-01

Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms. We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model. The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A' and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA' and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities. This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Microbial community structure characteristics associated membrane fouling in A/O-MBR system.

Science.gov (United States)

Gao, Da-Wen; Wen, Zhi-Dan; Li, Bao; Liang, Hong

2014-02-01

The study demonstrated the potential relationship between microbial community structure and membrane fouling in an anoxic-oxic membrane bioreactor (A/O-MBR). The results showed that the microbial community structure in biocake was different with aerobic mixture, and the dominant populations were out of sync during the fouling process. Based on microbial community structure and metabolites analysis, the results showed that the succession of microbial community might be the leading factor to the variation of metabolites, and it might be the primary cause of membrane fouling. The rise of Shannon diversity index (H) of the microbial community in A/O-MBR went with the gradually serious membrane fouling. Pareto-Lorenz curve was used to describe the evenness of microbial distribution in A/O-MBR, and the result indicated when community evenness was low, the membrane fouling took place smoothly or slightly, otherwise, high evenness of microbial community would lead to more seriously membrane fouling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Membrane-Dependent Effects of a Cytoplasmic Helix on the Structure and Drug Binding of the Influenza Virus M2 Protein

Science.gov (United States)

Cady, Sarah; Wang, Tuo; Hong, Mei

2011-01-01

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22 to 46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45 to 62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21–61). 13C-2H distance measurements of 13C-labeled protein and 2H-labeled amantadine showed that in DMPC bilayers, the first equivalent of drug bound S31 inside the M2(21–61) pore, similar to the behavior of M2TM in DMPC bilayers. The non-specific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21–61) is S31 in the TM pore. Interestingly, when M2(21–61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, while M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state, but did not rescue drug binding to M2(21–61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helices to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein. PMID:21661724

Probing Induced Structural Changes in Biomimetic Bacterial Cell Membrane Interactions with Divalent Cations

Energy Technology Data Exchange (ETDEWEB)

Holt, Allison M [ORNL; Standaert, Robert F [ORNL; Jubb, Aaron M [ORNL; Katsaras, John [ORNL; Johs, Alexander [ORNL

2017-01-01

Biological membranes, formed primarily by the self-assembly of complex mixtures of phospholipids, provide a structured scaffold for compartmentalization and structural processes in living cells. The specific physical properties of phospholipid species present in a given membrane play a key role in mediating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipid present in bacterial, yeast, and mammalian cell membranes, is exceptional. In addition to undergoing the standard lipid polymorphic transition between the gel and liquid-crystalline phase, it can also assume an unusual polymorphic state, the inverse hexagonal phase (HII). Divalent cations are among the factors that drive the formation of the HII phase, wherein the lipid molecules form stacked tubular structures by burying the hydrophilic head groups and exposing the hydrophobic tails to the bulk solvent. Most biological membranes contain a lipid species capable of forming the HII state suggesting that such lipid polymorphic structural states play an important role in structural biological processes such as membrane fusion. In this study, the interactions between Mg2+ and biomimetic bacterial cell membranes composed of PE and phosphatidylglycerol (PG) were probed using differential scanning calorimetry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectroscopy. The lipid phase transitions were examined at varying ratios of PE to PG and upon exposure to physiologically relevant concentrations of Mg2+. An understanding of these basic interactions enhances our understanding of membrane dynamics and how membrane-mediated structural changes may occur in vivo.

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

International Nuclear Information System (INIS)

Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

1990-01-01

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

Structuring detergents for extracting and stabilizing functional membrane proteins.

Directory of Open Access Journals (Sweden)

Rima Matar-Merheb

Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

Crystal structure of the plasma membrane proton pump

DEFF Research Database (Denmark)

Pedersen, Bjørn P.; Buch-Pedersen, Morten Jeppe; Morth, J. Preben

2007-01-01

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H1-ATPase (the proton pump) in plants and fungi1......-3, and Na1,K1-ATPase (the sodium-potassium pump) in animals4. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis5.The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na1,K1-ATPase and Ca21......- ATPase are type II6. Electron microscopy has revealed the overall shape of proton pumps7, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define...

Structure-based membrane dome mechanism for Piezo mechanosensitivity.

Science.gov (United States)

Guo, Yusong R; MacKinnon, Roderick

2017-12-12

Mechanosensitive ion channels convert external mechanical stimuli into electrochemical signals for critical processes including touch sensation, balance, and cardiovascular regulation. The best understood mechanosensitive channel, MscL, opens a wide pore, which accounts for mechanosensitive gating due to in-plane area expansion. Eukaryotic Piezo channels have a narrow pore and therefore must capture mechanical forces to control gating in another way. We present a cryo-EM structure of mouse Piezo1 in a closed conformation at 3.7Å-resolution. The channel is a triskelion with arms consisting of repeated arrays of 4-TM structural units surrounding a pore. Its shape deforms the membrane locally into a dome. We present a hypothesis in which the membrane deformation changes upon channel opening. Quantitatively, membrane tension will alter gating energetics in proportion to the change in projected area under the dome. This mechanism can account for highly sensitive mechanical gating in the setting of a narrow, cation-selective pore. © 2017, Guo et al.

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

Science.gov (United States)

Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

2017-08-01

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

The synthesis of recombinant membrane proteins in yeast for structural studies.

Science.gov (United States)

Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

2016-02-15

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Magnetic apatite for structural insights on the plasma membrane

International Nuclear Information System (INIS)

Stanca, Sarmiza E; Müller, Robert; Dellith, Jan; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph

2015-01-01

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications. (paper)

Magnetic apatite for structural insights on the plasma membrane

Science.gov (United States)

Stanca, Sarmiza E.; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

2015-01-01

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

Directory of Open Access Journals (Sweden)

Jose L S Lopes

Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia.

Science.gov (United States)

Pizarro-Cerdá, Javier; Charbit, Alain; Enninga, Jost; Lafont, Frank; Cossart, Pascale

2016-12-01

Bacterial pathogens display an impressive arsenal of molecular mechanisms that allow survival in diverse host niches. Subversion of plasma membrane and cytoskeletal functions are common themes associated to infection by both extracellular and intracellular pathogens. Moreover, intracellular pathogens modify the structure/stability of their membrane-bound compartments and escape degradation from phagocytic or autophagic pathways. Here, we review the manipulation of host membranes by Listeria monocytogenes, Francisella tularensis, Shigella flexneri and Yersinia spp. These four bacterial model pathogens exemplify generalized strategies as well as specific features observed during bacterial infection processes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cell wall-bound silicon optimizes ammonium uptake and metabolism in rice cells.

Science.gov (United States)

Sheng, Huachun; Ma, Jie; Pu, Junbao; Wang, Lijun

2018-05-16

Turgor-driven plant cell growth depends on cell wall structure and mechanics. Strengthening of cell walls on the basis of an association and interaction with silicon (Si) could lead to improved nutrient uptake and optimized growth and metabolism in rice (Oryza sativa). However, the structural basis and physiological mechanisms of nutrient uptake and metabolism optimization under Si assistance remain obscure. Single-cell level biophysical measurements, including in situ non-invasive micro-testing (NMT) of NH4+ ion fluxes, atomic force microscopy (AFM) of cell walls, and electrolyte leakage and membrane potential, as well as whole-cell proteomics using isobaric tags for relative and absolute quantification (iTRAQ), were performed. The altered cell wall structure increases the uptake rate of the main nutrient NH4+ in Si-accumulating cells, whereas the rate is only half in Si-deprived counterparts. Rigid cell walls enhanced by a wall-bound form of Si as the structural basis stabilize cell membranes. This, in turn, optimizes nutrient uptake of the cells in the same growth phase without any requirement for up-regulation of transmembrane ammonium transporters. Optimization of cellular nutrient acquisition strategies can substantially improve performance in terms of growth, metabolism and stress resistance.

Structurally stable graphene oxide-based nanofiltration membranes with bioadhesive polydopamine coating

Science.gov (United States)

Wang, Chongbin; Li, Zhiyuan; Chen, Jianxin; Yin, Yongheng; Wu, Hong

2018-01-01

Graphene oxide (GO)-based membranes possess promising potential in liquid separation for its high flux. The state-of-art GO-based membranes need to be supported by a substrate to ensure that the ultra-thin GO layer can withstand transmembrane pressure in practical applications. The interfacial compatibility of this kind of composite membrane remains a great challenge due to the intrinsic difference in chemical/physical properties between the GO sheets and the substrate. In this paper, a structurally stable GO-based composite nanofiltration membrane was fabricated by coupling the mussel-inspired adhesive platform and filtration-assisted assembly of GO laminates. The water flux for the prepared GO-based nanofiltration membrane reached up to 85 L m-2 h-1 bar-1 with a high retention above 95% and 100% for Orange G and Congo Red, respectively. The membrane exhibited highly stable structure owing to the covalent and noncovalent interactions between GO separation layer and dopamine adhesive platform.

The performance of double layer structure membrane prepared from flowing coagulant

Science.gov (United States)

Mieow Kee, Chan; Xeng, Anthony Leong Chan; Regal, Sasiskala; Singh, Balvinder; Raoo, Preeshaath; Koon Eu, Yap; Sok Choo, Ng

2017-12-01

Membrane with double layer structure is favourable as it exhibits smooth surface and macrovoids free structure. However, its’ performance in terms of permeability, porosity and strength has not been studied thoroughly. Additionally, the effect of flowing coagulant on the formation of double layer membrane has not been reported. Thus, the objective of this study is to investigate the performance of double layer membranes, which were prepared using flowing coagulant. Results showed that when the coagulant flow changed from laminar to turbulent, the pure water permeation of the membrane increased. It was due to the higher porosity in the membrane, which prepared by turbulent flow (CA-Turbulent) compared to the membrane which fabricated under laminar condition (CA-Laminar). This can be explained by the rapid solvent-coagulant exchange rate between the polymer solution and the turbulent coagulant. In term of strength, the tensile strength of the CA-Turbulent was ~32 MPa, which was 100% higher compared to CA-Laminar. This may due to the presence of large amount of nodules on its surface, which reduced the surface integrity. In conclusion, flowing coagulant altered the membrane properties and adopting turbulent coagulant flow in membrane fabrication would improve the porosity, surface roughness and the strength of the membrane.

Impacts of operating conditions and solution chemistry on osmotic membrane structure and performance

KAUST Repository

Wong, Mavis C.Y.

2012-02-01

Herein, we report on changes in the performance of a commercial cellulose triacetate (CTA) membrane, imparted by varied operating conditions and solution chemistries. Changes to feed and draw solution flow rate did not significantly alter the CTA membrane\\'s water permeability, salt permeability, or membrane structural parameter when operated with the membrane skin layer facing the draw solution (PRO-mode). However, water and salt permeability increased with increasing feed or draw solution temperature, while the membrane structural parameter decreased with increasing draw solution, possibly due to changes in polymer intermolecular interactions. High ionic strength draw solutions may de-swell the CTA membrane via charge neutralization, which resulted in lower water permeability, higher salt permeability, and lower structural parameter. This observed trend was further exacerbated by the presence of divalent cations which tends to swell the polymer to a greater extent. Finally, the calculated CTA membrane\\'s structural parameter was lower and less sensitive to external factors when operated in PRO-mode, but highly sensitive to the same factors when the skin layer faced the feed solution (FO-mode), presumably due to swelling/de-swelling of the saturated porous substructure by the draw solution. This is a first attempt aimed at systematically evaluating the changes in performance of the CTA membrane due to operating conditions and solution chemistry, shedding new insight into the possible advantages and disadvantages of this material in certain applications. © 2011 Elsevier B.V.

Increasing the Performance of Vacuum Membrane Distillation Using Micro-Structured Hydrophobic Aluminum Hollow Fiber Membranes

Directory of Open Access Journals (Sweden)

Chia-Chieh Ko

2017-04-01

Full Text Available This study develops a micro-structured hydrophobic alumina hollow fiber with a high permeate flux of 60 Lm−2h−1 and salt rejection over 99.9% in a vacuum membrane distillation process. The fiber is fabricated by phase inversion and sintering, and then modified with fluoroalkylsilanes to render it hydrophobic. The influence of the sintering temperature and feeding temperature in membrane distillation (MD on the characteristics of the fiber and MD performance are investigated. The vacuum membrane distillation uses 3.5 wt % NaCl aqueous solution at 70 °C at 0.03 bar. The permeate flux of 60 Lm−2h−1 is the highest, compared with reported data and is higher than that for polymeric hollow fiber membranes.

Structural and morphological changes in supramolecular-structured polymer electrolyte membrane fuel cell on addition of phosphoric acid

Science.gov (United States)

Hendrana, S.; Pryliana, R. F.; Natanael, C. L.; Rahayu, I.

2018-03-01

Phosphoric acid is one agents used in membrane fuel cell to modify ionic conductivity. Therefore, its distribution in membrane is a key parameter to gain expected conductivity. Efforts have been made to distribute phosphoric acid in a supramolecular-structured membrane prepared with a matrix. To achieve even distribution across bulk of the membrane, the inclusion of the polyacid is carried out under pressurized chamber. Image of scanning electron microscopy (SEM) shows better phosphoric acid distribution for one prepared in pressurized state. It also leads in better performing in ionic conductivity. Moreover, data from differential scanning calorimetry (DSC) indicate that the addition of phosphoric acid is prominent in the change of membrane structure, while morphological changes are captured in SEM images.

Discovery of novel membrane binding structures and functions

Science.gov (United States)

Kufareva, Irina; Lenoir, Marc; Dancea, Felician; Sridhar, Pooja; Raush, Eugene; Bissig, Christin; Gruenberg, Jean; Abagyan, Ruben; Overduin, Michael

2014-01-01

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms, and could uncover novel sites for therapeutic intervention. Here we present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH) and PX domains bind membranes, the resulting Membrane Optimal Docking Area (MODA) method yields predictions for a given protein of known three dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain and Neisseria gonorrhoeae MsrB protein, and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR) which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible, and provides a new tool for functional annotation of the proteome. PMID:25394204

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Homogenization-based interval analysis for structural-acoustic problem involving periodical composites and multi-scale uncertain-but-bounded parameters.

Science.gov (United States)

Chen, Ning; Yu, Dejie; Xia, Baizhan; Liu, Jian; Ma, Zhengdong

2017-04-01

This paper presents a homogenization-based interval analysis method for the prediction of coupled structural-acoustic systems involving periodical composites and multi-scale uncertain-but-bounded parameters. In the structural-acoustic system, the macro plate structure is assumed to be composed of a periodically uniform microstructure. The equivalent macro material properties of the microstructure are computed using the homogenization method. By integrating the first-order Taylor expansion interval analysis method with the homogenization-based finite element method, a homogenization-based interval finite element method (HIFEM) is developed to solve a periodical composite structural-acoustic system with multi-scale uncertain-but-bounded parameters. The corresponding formulations of the HIFEM are deduced. A subinterval technique is also introduced into the HIFEM for higher accuracy. Numerical examples of a hexahedral box and an automobile passenger compartment are given to demonstrate the efficiency of the presented method for a periodical composite structural-acoustic system with multi-scale uncertain-but-bounded parameters.

Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

DEFF Research Database (Denmark)

Link, Todd M; Valentin-Hansen, Poul; Brennan, Richard G

2009-01-01

(A) RNA, A(15). The structure reveals a unique RNA binding mechanism. Unlike uridine-containing sequences, which bind to the "proximal" face, the poly(A) tract binds to the "distal" face of Hfq using 6 tripartite binding motifs. Each motif consists of an adenosine specificity site (A site), which......Hfq is a small, highly abundant hexameric protein that is found in many bacteria and plays a critical role in mRNA expression and RNA stability. As an "RNA chaperone," Hfq binds AU-rich sequences and facilitates the trans annealing of small RNAs (sRNAs) to their target mRNAs, typically resulting...... in the down-regulation of gene expression. Hfq also plays a key role in bacterial RNA decay by binding tightly to polyadenylate [poly(A)] tracts. The structural mechanism by which Hfq recognizes and binds poly(A) is unknown. Here, we report the crystal structure of Escherichia coli Hfq bound to the poly...

Solution structure and elevator mechanism of the membrane electron transporter CcdA.

Science.gov (United States)

Zhou, Yunpeng; Bushweller, John H

2018-02-01

Membrane oxidoreductase CcdA plays a central role in supplying reducing equivalents from the bacterial cytoplasm to the envelope. It transports electrons across the membrane using a single pair of cysteines by a mechanism that has not yet been elucidated. Here we report an NMR structure of the Thermus thermophilus CcdA (TtCcdA) in an oxidized and outward-facing state. CcdA consists of two inverted structural repeats of three transmembrane helices (2 × 3-TM). We computationally modeled and experimentally validated an inward-facing state, which suggests that CcdA uses an elevator-type movement to shuttle the reactive cysteines across the membrane. CcdA belongs to the LysE superfamily, and thus its structure may be relevant to other LysE clan transporters. Structure comparisons of CcdA, semiSWEET, Pnu, and major facilitator superfamily (MFS) transporters provide insights into membrane transporter architecture and mechanism.

Structure of V-type ATPase from Clostridium fervidus by electron microscopy

NARCIS (Netherlands)

Boekema, EJ; Ubbink-Kok, T; Lolkema, JS; Brisson, A; Konings, WN

F-type and V-type ATPases couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes and have similarities in structure and mechanism. In both types of enzymes three main parts can be distinguished: headpiece, membrane-bound piece and stalk region. We report

Identification of soil bacteria able to degrade phenanthrene bound to a hydrophobic sorbent in situ

Energy Technology Data Exchange (ETDEWEB)

Regonne, Raïssa Kom [CEA, DSV/iRTSV, Chimie et Biologie des Métaux, 38054, Grenoble cedex 9 (France); Univ. Grenoble Alpes and CNRS, UMR 5249, 38042, Grenoble (France); Laboratoire de Substances Actives et Pollution, ENSAI, Université de Ngaoundéré, BP 455, Ngaoundéré (Cameroon); Martin, Florence [CEA, DSV/iRTSV, Chimie et Biologie des Métaux, 38054, Grenoble cedex 9 (France); Univ. Grenoble Alpes and CNRS, UMR 5249, 38042, Grenoble (France); Mbawala, Augustin [Laboratoire de Microbiologie, ENSAI, Université de Ngaoundéré, BP 455, Ngaoundéré (Cameroon); Ngassoum, Martin Benoît [Laboratoire de Substances Actives et Pollution, ENSAI, Université de Ngaoundéré, BP 455, Ngaoundéré (Cameroon); Jouanneau, Yves [CEA, DSV/iRTSV, Chimie et Biologie des Métaux, 38054, Grenoble cedex 9 (France); Univ. Grenoble Alpes and CNRS, UMR 5249, 38042, Grenoble (France)

2013-09-15

Efficient bioremediation of PAH-contaminated sites is limited by the hydrophobic character and poor bioavailability of pollutants. In this study, stable isotope probing (SIP) was implemented to track bacteria that can degrade PAHs adsorbed on hydrophobic sorbents. Temperate and tropical soils were incubated with {sup 13}C-labeled phenanthrene, supplied by spiking or coated onto membranes. Phenanthrene mineralization was faster in microcosms with PAH-coated membranes than in microcosms containing spiked soil. Upon incubation with temperate soil, phenanthrene degraders found in the biofilms that formed on coated membranes were mainly identified as Sphingomonadaceae and Actinobacteria. In the tropical soil, uncultured Rhodocyclaceae dominated degraders bound to membranes. Accordingly, ring-hydroxylating dioxygenase sequences recovered from this soil matched PAH-specific dioxygenase genes recently found in Rhodocyclaceae. Hence, our SIP approach allowed the detection of novel degraders, mostly uncultured, which differ from those detected after soil spiking, but might play a key role in the bioremediation of PAH-polluted soils. -- Highlights: •Soil bacteria with the ability to degrade sorbent-bound PAHs were investigated. •In soil, membrane-bound phenanthrene was readily mineralized. •PAH degraders found in biofilms were different in temperate and tropical soils. •Uncultured Rhodocyclaceae were dominant phenanthrene degraders in the tropical soil. •PAH-specific ring-hydroxylating dioxygenase sequences were identified in soil DNA. -- Bacteria able to degrade PAHs bound to a hydrophobic sorbent were mainly identified as uncultured Rhodocyclaceae and Sphingomonadaceae in polluted soils from tropical and temperate area, respectively.

Identification of soil bacteria able to degrade phenanthrene bound to a hydrophobic sorbent in situ

International Nuclear Information System (INIS)

Regonne, Raïssa Kom; Martin, Florence; Mbawala, Augustin; Ngassoum, Martin Benoît; Jouanneau, Yves

2013-01-01

Efficient bioremediation of PAH-contaminated sites is limited by the hydrophobic character and poor bioavailability of pollutants. In this study, stable isotope probing (SIP) was implemented to track bacteria that can degrade PAHs adsorbed on hydrophobic sorbents. Temperate and tropical soils were incubated with 13 C-labeled phenanthrene, supplied by spiking or coated onto membranes. Phenanthrene mineralization was faster in microcosms with PAH-coated membranes than in microcosms containing spiked soil. Upon incubation with temperate soil, phenanthrene degraders found in the biofilms that formed on coated membranes were mainly identified as Sphingomonadaceae and Actinobacteria. In the tropical soil, uncultured Rhodocyclaceae dominated degraders bound to membranes. Accordingly, ring-hydroxylating dioxygenase sequences recovered from this soil matched PAH-specific dioxygenase genes recently found in Rhodocyclaceae. Hence, our SIP approach allowed the detection of novel degraders, mostly uncultured, which differ from those detected after soil spiking, but might play a key role in the bioremediation of PAH-polluted soils. -- Highlights: •Soil bacteria with the ability to degrade sorbent-bound PAHs were investigated. •In soil, membrane-bound phenanthrene was readily mineralized. •PAH degraders found in biofilms were different in temperate and tropical soils. •Uncultured Rhodocyclaceae were dominant phenanthrene degraders in the tropical soil. •PAH-specific ring-hydroxylating dioxygenase sequences were identified in soil DNA. -- Bacteria able to degrade PAHs bound to a hydrophobic sorbent were mainly identified as uncultured Rhodocyclaceae and Sphingomonadaceae in polluted soils from tropical and temperate area, respectively

Influence of the Relative Enamel Abrasivity (REA) of Toothpastes on the Uptake of KOH-soluble and Structurally Bound Fluoride.

Science.gov (United States)

Elmazi, Valbona; Sener, Beatrice; Attin, Thomas; Imfeld, Thomas; Wegehaupt, Florian J

2015-01-01

To determine the influence of the relative enamel abrasivity (REA) of fluoridated toothpaste on the uptake of KOH-soluble and structurally bound fluoride into enamel. Bovine enamel samples were randomly allocated to 6 groups (n=36 per group). Groups A to C were treated with sodium fluoride (NaF) toothpastes and groups D to F with amine fluoride (AmF) toothpastes (1500 ppm F each). The REA in groups A and D was 2, in groups B and E it was 6 and in groups C and F it was 9. Twice a day, 18 samples of each group were immersed for 2 min in a slurry (toothpaste:artificial saliva=1:3), while the remaining samples were brushed with the respective slurry (2.5 N force; 60 strokes/min; 2 min). All samples were stored at 37°C and 100% humidity. After five days, the amount of KOH-soluble and structurally bound fluoride was determined and statistically compared by Scheffe's post-hoc tests. REA value and mode of application (immersion or brushing) had no significant influence on the amount of either kind of fluoride from NaF toothpastes. Only for the NaF toothpaste with REA 6 was the amount of KOH-soluble fluoride significantly higher after brushing. With AmF toothpastes, KOH-soluble and structurally bound fluoride concentrations were significantly higher when the samples were brushed. Furthermore, in the REA-2 group, the amounts of KOH-soluble fluoride (brushed or immersed) and structurally bound fluoride (brushed) were significantly higher than in the other groups. The REA dependency of KOH-soluble and structurally bound fluoride was found only for the AmF toothpastes. Using AmF toothpaste, the mode of application influenced the uptake of KOH-soluble and structurally bound fluoride into enamel.

Toward the Structure of Dynamic Membrane-Anchored Actin Networks

Science.gov (United States)

Weber, Igor

2007-01-01

In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

Overcoming barriers to membrane protein structure determination

NARCIS (Netherlands)

Bill, Roslyn M.; Henderson, Peter J. F.; Iwata, So; Kunji, Edmund R. S.; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G.; Vogel, Horst

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new

Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

Science.gov (United States)

Pohorille, Andrew

2003-01-01

Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4

Isolation and characterization of MUC15, a novel cell membrane-associated mucin

DEFF Research Database (Denmark)

Pallesen, Lone Tjener; Berglund, Lars; Rasmussen, Lone Kjær

2002-01-01

The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length c......-like protein was named MUC15 by appointment of the HUGO Gene Nomenclature Committee. The deduced amino-acid sequences of human and bovine MUC15 demonstrated structural hallmarks characteristic for other membrane-bound mucins, such as a serine, threonine, and proline-rich extracellular region with several...

Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs

Energy Technology Data Exchange (ETDEWEB)

Hagn, Franz, E-mail: franz.hagn@tum.de; Wagner, Gerhard, E-mail: gerhard-wagner@hms.harvard.edu [Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology (United States)

2015-04-15

NMR structural studies on membrane proteins are often complicated by their large size, taking into account the contribution of the membrane mimetic. Therefore, classical resonance assignment approaches often fail. The large size of phospholipid nanodiscs, a detergent-free phospholipid bilayer mimetic, prevented their use in high-resolution solution-state NMR spectroscopy so far. We recently introduced smaller nanodiscs that are suitable for NMR structure determination. However, side-chain assignments of a membrane protein in nanodiscs still remain elusive. Here, we utilized a NOE-based approach to assign (stereo-) specifically labeled Ile, Leu, Val and Ala methyl labeled and uniformly {sup 15}N-Phe and {sup 15}N-Tyr labeled OmpX and calculated a refined high-resolution structure. In addition, we were able to obtain residual dipolar couplings (RDCs) of OmpX in nanodiscs using Pf1 phage medium for the induction of weak alignment. Back-calculated NOESY spectra of the obtained NMR structures were compared to experimental NOESYs in order to validate the quality of these structures. We further used NOE information between protonated lipid head groups and side-chain methyls to determine the position of OmpX in the phospholipid bilayer. These data were verified by paramagnetic relaxation enhancement (PRE) experiments obtained with Gd{sup 3+}-modified lipids. Taken together, this study emphasizes the need for the (stereo-) specific labeling of membrane proteins in a highly deuterated background for high-resolution structure determination, particularly in large membrane mimicking systems like phospholipid nanodiscs. Structure validation by NOESY back-calculation will be helpful for the structure determination and validation of membrane proteins where NOE assignment is often difficult. The use of protein to lipid NOEs will be beneficial for the positioning of a membrane protein in the lipid bilayer without the need for preparing multiple protein samples.

Synthetic membrane-targeted antibiotics.

Science.gov (United States)

Vooturi, S K; Firestine, S M

2010-01-01

Antimicrobial resistance continues to evolve and presents serious challenges in the therapy of both nosocomial and community-acquired infections. The rise of resistant strains like methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-resistant enterococci (VRE) suggests that antimicrobial resistance is an inevitable evolutionary response to antimicrobial use. This highlights the tremendous need for antibiotics against new bacterial targets. Agents that target the integrity of bacterial membrane are relatively novel in the clinical armamentarium. Daptomycin, a lipopeptide is a classical example of membrane-bound antibiotic. Nature has also utilized this tactic. Antimicrobial peptides (AMPs), which are found in all kingdoms, function primarily by permeabilizing the bacterial membrane. AMPs have several advantages over existing antibiotics including a broad spectrum of activity, rapid bactericidal activity, no cross-resistance with the existing antibiotics and a low probability for developing resistance. Currently, a small number of peptides have been developed for clinical use but therapeutic applications are limited because of poor bioavailability and high manufacturing cost. However, their broad specificity, potent activity and lower probability for resistance have spurred the search for synthetic mimetics of antimicrobial peptides as membrane-active antibiotics. In this review, we will discuss the different classes of synthetic membrane-bound antibiotics published since 2004.

Formation of oriented membrane multilayers of Na/K-ATPase

International Nuclear Information System (INIS)

Pachence, J.M.; Knott, R.; Edelman, I.S.; Schoenborn, B.P.; Wallace, B.A.

1982-01-01

The isolated membrane-bound enzyme retains its ouabain-sensitive ATP hydrolysis activity, and produces ATP-dependent Na + and K + fluxes when incorporated into phospholipid vesicles. The ultimate goal of this work is to determine its low resolution structure using both X-ray and neutron diffraction. A number of methods were used to impart lamellar stacking order to highly purified pig Na/K-ATPase membranes. Upon partial dehydration, x-ray diffraction from Na/K-ATPase membrane multilayers at 98% relative humidity yielded discrete reflections of 118 A periodicity, diffracting to 1/14.8 A -1 , additionally, continuous diffraction to 1/10 A -1 was obtained. Subjecting the membrane multilayers to high magnetic fields improved the quality of the lamellar diffraction dramatically. Neutron diffraction studies of the partially dehydrated Na/K-ATPase membrane multilayers detected a mosaic spread of 2 0 when the samples were subjected to a magnetic field of 5 Tesla perpendicular to the membrane surface; the reflections were narrower than the camera line width; hence, the lattice disorder has also decreased significantly, although only four orders were measured

Structural Changes of PVDF Membranes by Phase Separation Control

International Nuclear Information System (INIS)

Lee, Semin; Kim, Sung Soo

2016-01-01

Thermally induced phase separation (TIPS) and nonsolvent induced phase separation (NIPS) were simultaneously induced for the preparation of flat PVDF membranes. N-methyl-2-pyrrolidone (NMP) was used as a solvent and dibutyl-phthlate (DBP) was used as a diluent for PVDF. When PVDF was melt blended with NMP and DBP, crystallization temperature was lowered for TIPS and unstable region was expanded for NIPS. Ratio of solvent to diluent changed the phase separation mechanism to obtain the various membrane structures. Contact mode of dope solution with nonsolvent determined the dominant phase separation behavior. Since heat transfer rate was greater than mass transfer rate, surface structure was formed by NIPS and inner structure was by TIPS. Quenching temperature of dope solution also affected the phase separation mechanism and phase separation rate to result in the variation of structure

Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

Directory of Open Access Journals (Sweden)

Jie Chen

Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

Science.gov (United States)

Lei, Li; Egli, Martin

2016-04-01

Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

Dielectric structures with bound modes for microcavity lasers

NARCIS (Netherlands)

Visser, P.M.; Allaart, K.; Lenstra, D.

2002-01-01

Cavity modes of dielectric microsphcres and vertical cavity surface emitting lasers, in spite of their high Q, are never exactly bound, but have a finite width due to leakage at the borders. We propose types of microstructures that sustain three-dimensionally bound modes of the radiation field when

Super-resolution optical microscopy for studying membrane structure and dynamics.

Science.gov (United States)

Sezgin, Erdinc

2017-07-12

Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

International Nuclear Information System (INIS)

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

2006-01-01

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

Structural and electrical characterization of PZT on gold for micromachined piezoelectric membranes

International Nuclear Information System (INIS)

Robinson, M.C.; Morris, D.J.; Hayenga, P.D.; Cho, J.H.; Richards, C.D.; Richards, R.F.; Bahr, D.F.

2006-01-01

Piezoelectric membranes have been fabricated that incorporate a gold bottom electrode with an adhesion layer of titanium-tungsten (10:90 wt. %). For solution-deposited acetic acid based lead zirconate titanate (HoAc-PZT) with a Zr:Ti ratio of 40:60, the film's average piezoelectric coefficient, e 31 , is -5.31 C/m 2 , with a dielectric constant of 814 at 200 Hz, which is similar to values for platinum bottom electrodes. The PZT structure remains columnar on both types of bottom electrodes. Initial fabrication attempts resulted in cracking that initiated in the PZT layer of the structure. X-ray photoelectron spectroscopy was utilized to establish how processing affects diffusion throughout the composite membrane structure. Crack-free membranes were fabricated and tested. This paper discusses the performance properties and piezoelectric fatigue results for these membranes. (orig.)

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering

DEFF Research Database (Denmark)

Midtgaard, Søren Roi; Darwish, Tamim A.; Pedersen, Martin Cramer

2018-01-01

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron sca...... solution structure determination of membrane proteins by SANS and subsequent data analysis available to non-specialists. This article is protected by copyright. All rights reserved....

The in vivo structure of biological membranes and evidence for lipid domains

Energy Technology Data Exchange (ETDEWEB)

Nickels, Jonathan D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Chatterjee, Sneha [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Stanley, Christopher B. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Qian, Shuo [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Cheng, Xiaolin [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Myles, Dean A. A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Standaert, Robert F. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Elkins, James G. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Katsaras, John [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Lopez, Daniel [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)

2017-05-23

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments—performed under biologically relevant conditions—answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.

Impacts of operating conditions and solution chemistry on osmotic membrane structure and performance

KAUST Repository

Wong, Mavis C.Y.; Martinez, Kristina; Ramon, Guy Z.; Hoek, Eric M.V.

2012-01-01

Herein, we report on changes in the performance of a commercial cellulose triacetate (CTA) membrane, imparted by varied operating conditions and solution chemistries. Changes to feed and draw solution flow rate did not significantly alter the CTA membrane's water permeability, salt permeability, or membrane structural parameter when operated with the membrane skin layer facing the draw solution (PRO-mode). However, water and salt permeability increased with increasing feed or draw solution temperature, while the membrane structural parameter decreased with increasing draw solution, possibly due to changes in polymer intermolecular interactions. High ionic strength draw solutions may de-swell the CTA membrane via charge neutralization, which resulted in lower water permeability, higher salt permeability, and lower structural parameter. This observed trend was further exacerbated by the presence of divalent cations which tends to swell the polymer to a greater extent. Finally, the calculated CTA membrane's structural parameter was lower and less sensitive to external factors when operated in PRO-mode, but highly sensitive to the same factors when the skin layer faced the feed solution (FO-mode), presumably due to swelling/de-swelling of the saturated porous substructure by the draw solution. This is a first attempt aimed at systematically evaluating the changes in performance of the CTA membrane due to operating conditions and solution chemistry, shedding new insight into the possible advantages and disadvantages of this material in certain applications. © 2011 Elsevier B.V.

Fish skin as a model membrane: structure and characteristics.

Science.gov (United States)

Konrádsdóttir, Fífa; Loftsson, Thorsteinn; Sigfússon, Sigurdur Dadi

2009-01-01

Synthetic and cell-based membranes are frequently used during drug formulation development for the assessment of drug availability. However, most of the currently used membranes do not mimic mucosal membranes well, especially the aqueous mucous layer of the membranes. In this study we evaluated catfish (Anarichas lupus L) skin as a model membrane. Permeation of hydrocortisone, lidocaine hydrochloride, benzocaine, diethylstilbestrol, naproxen, picric acid and sodium nitrate through skin from a freshly caught catfish was determined in Franz diffusion cells. Both lipophilic and hydrophilic molecules permeate through catfish skin via hydrated channels or aqueous pores. No correlation was observed between the octanol/water partition coefficient of the permeating molecules and their permeability coefficient through the skin. Permeation through catfish skin was found to be diffusion controlled. The results suggest that permeation through the fish skin proceeds via a diffusion-controlled process, a process that is similar to drug permeation through the aqueous mucous layer of a mucosal membrane. In addition, the fish skin, with its collagen matrix structure, appears to possess similar properties to the eye sclera.

Fast iodide-SAD phasing for high-throughput membrane protein structure determination.

Science.gov (United States)

Melnikov, Igor; Polovinkin, Vitaly; Kovalev, Kirill; Gushchin, Ivan; Shevtsov, Mikhail; Shevchenko, Vitaly; Mishin, Alexey; Alekseev, Alexey; Rodriguez-Valera, Francisco; Borshchevskiy, Valentin; Cherezov, Vadim; Leonard, Gordon A; Gordeliy, Valentin; Popov, Alexander

2017-05-01

We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide-single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins-the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein-coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques.

Influence of ionizing radiation on the spatial structure of erythrocyte membranes

International Nuclear Information System (INIS)

Dreval', V.Yi.; Syichevs'ka, L.V.; Doroshenko, A.O.; Roshal', O.D.

1998-01-01

Influence of gamma-radiation of doses of 10, 10 2 , 5 centre dot 10 2 , and 10 3 Gy on the structure of the protein-lipid complexes of erythrocyte membranes is investigated. The allotment of fluorescence of protein in the donor-acceptor pair of tryptophan-pyrene and the distance of protein from the surface of the lipid bilayer of a membrane are determined by the method of inductive-resonance transfer of energy. The pair is localized at the distance of above 3.2 nm from lipids. We find that the action of irradiation changes the space structure of proteins and lipids of the erythrocyte membrane

Structural basis for catalysis at the membrane-water interface.

Science.gov (United States)

Dufrisne, Meagan Belcher; Petrou, Vasileios I; Clarke, Oliver B; Mancia, Filippo

2017-11-01

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Robust hydrophobic polyurethane fibrous membranes with tunable porous structure for waterproof and breathable application

Science.gov (United States)

Gu, Jiatai; Gu, Haihong; Cao, Jin; Chen, Shaojie; Li, Ni; Xiong, Jie

2018-05-01

In this work, novel nanofibrous membranes with waterproof and breathable (W&B) performance were successfully fabricated by the combination of electrospinning and surface modification technology. This fibrous membranes consisted of polyurethane (PU), NaCl, and fluoroalkylsilane (FAS). Firstly, The fibrous construction and porous structure of fibrous membranes were regulated by tuning the NaCl concentrations in PU solutions. Then, the obtained PU/NaCl fibrous membranes were further modified with fluoroalkylsilane (FAS) to improve hydrophobic property. The synergistic effect of porous structure and hydrophobicity on waterproof and breathable performance was investigated. Furthermore, the mechanical property of fibrous membranes was deeply analysed on the basis of macromolecule orientation and adhesive structure. Benefiting from the optimized porous structure and hydrophobic modification, the resultant fibrous membranes exhibited excellent waterproof (hydrostatic pressure of 1261 Mbar), breathable (water vapor transmission (WVT) rate of 9.06 kg m-2 d-1 and air permeability of 4.8 mm s-1) performance, as well as high tensile strength (breakage stress of 10.4 MPa), suggesting a promising candidate for various applications, especially in protective clothing.

Multifunctional-layered materials for creating membrane-restricted nanodomains and nanoscale imaging

Energy Technology Data Exchange (ETDEWEB)

Srinivasan, P., E-mail: prasri@ece.ucsb.edu, E-mail: srinivasan@lifesci.ucsb.edu [Department of Electrical and Computer Engineering, University of California, Santa Barbara, California 93106, USA and Neuroscience Research Institute, University of California, Santa Barbara, California 93106 (United States)

2016-01-18

Experimental platform that allows precise spatial positioning of biomolecules with an exquisite control at nanometer length scales is a valuable tool to study the molecular mechanisms of membrane bound signaling. Using micromachined thin film gold (Au) in layered architecture, it is possible to add both optical and biochemical functionalities in in vitro. Towards this goal, here, I show that docking of complementary DNA tethered giant phospholiposomes on Au surface can create membrane-restricted nanodomains. These nanodomains are critical features to dissect molecular choreography of membrane signaling complexes. The excited surface plasmon resonance modes of Au allow label-free imaging at diffraction-limited resolution of stably docked DNA tethered phospholiposomes, and lipid-detergent bicelle structures. Such multifunctional building block enables realizing rigorously controlled in vitro set-up to model membrane anchored biological signaling, besides serving as an optical tool for nanoscale imaging.

Measurement and evaluation of the summer microclimate in the semi-enclosed space under a membrane structure

Energy Technology Data Exchange (ETDEWEB)

He, Jiang; Hoyano, Akira [Department of Environmental Science and Technology, Interdisciplinary Graduate School, Tokyo Institute of Technology, 4259-G5-2 Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan)

2010-01-15

This study aims to clarify the summer microclimate in membrane structure buildings with semi-outdoor spaces and develop a computational simulation tool for designing a comfortable urban environment using membrane structures. Field measurements were conducted in a membrane structure building with a semi-outdoor space during a summer period. The present paper describes analysis results of measurement data for vertical distributions of air temperature and velocity under the membrane structure on clear sunny days. The following subjects were also discussed: (1) the effect of solar transmission on the warming of air temperature by the floor under the membrane structure; (2) the temperature reduction effect of ventilation by wind; (3) evaluation of thermal comfort in the living space under the membrane structure in terms of a thermal comfort index (new standard effective temperature: SET*). In order to demonstrate the capability to improve the thermal environment in the test membrane structure building, an evaporative cooling pavement was assumed to be applied to the ground under the membrane structure. The microclimatic modifying effect of this passive cooling strategy was evaluated using a numerical simulation method of coupling computational fluid dynamics (CFD) with a 3D-CAD-based thermal simulation tool developed by the authors' research group. Simulation results show that the proposed simulation method is capable of quantifying spatial distributions of surface temperature, air temperature, air velocity and moisture in the living space under the membrane structure. The thermal comfort index (SET*) can also be estimated using these simulated results. (author)

Structure and organization of nanosized-inclusion-containing bilayer membranes

Science.gov (United States)

Ren, Chun-Lai; Ma, Yu-Qiang

2009-07-01

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

A Glimpse of Membrane Transport through Structures-Advances in the Structural Biology of the GLUT Glucose Transporters.

Science.gov (United States)

Yan, Nieng

2017-08-18

The cellular uptake of glucose is an essential physiological process, and movement of glucose across biological membranes requires specialized transporters. The major facilitator superfamily glucose transporters GLUTs, encoded by the SLC2A genes, have been a paradigm for functional, mechanistic, and structural understanding of solute transport in the past century. This review starts with a glimpse into the structural biology of membrane proteins and particularly membrane transport proteins, enumerating the landmark structures in the past 25years. The recent breakthrough in the structural elucidation of GLUTs is then elaborated following a brief overview of the research history of these archetypal transporters, their functional specificity, and physiological and pathophysiological significances. Structures of GLUT1, GLUT3, and GLUT5 in distinct transport and/or ligand-binding states reveal detailed mechanisms of the alternating access transport cycle and substrate recognition, and thus illuminate a path by which structure-based drug design may be applied to help discover novel therapeutics against several debilitating human diseases associated with GLUT malfunction and/or misregulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Non-cooperative immobilization of residual water bound in lyophilized photosynthetic lamellae.

Science.gov (United States)

Harańczyk, Hubert; Baran, Ewelina; Nowak, Piotr; Florek-Wojciechowska, Małgorzata; Leja, Anna; Zalitacz, Dorota; Strzałka, Kazimierz

2015-12-01

This study applied 1H-NMR in time and in frequency domain measurements to monitor the changes that occur in bound water dynamics at decreased temperature and with increased hydration level in lyophilizates of native wheat photosynthetic lamellae and in photosynthetic lamellae reconstituted from lyophilizate. Proton relaxometry (measured as free induction decay = FID) distinguishes a Gaussian component S within the NMR signal (o). This comes from protons of the solid matrix of the lamellae and consists of (i) an exponentially decaying contribution L1 from mobile membrane protons, presumably from lipids, and from water that is tightly bound to the membrane surface and thus restricted in mobility; and (ii) an exponentially decaying component L2 from more mobile, loosely bound water pool. Both proton relaxometry data and proton spectroscopy show that dry lyophilizate incubated in dry air, i.e., at a relative humidity (p/p0) of 0% reveals a relatively high hydration level. The observed liquid signal most likely originates from mobile membrane protons and a tightly bound water fraction that is sealed in pores of dry lyophilizate and thus restricted in mobility. The estimations suggest that the amount of sealed water does not exceed the value characteristic for the main hydration shell of a phospholipid. Proton spectra collected for dry lyophilizate of photosynthetic lamellae show a continuous decrease in the liquid signal component without a distinct freezing transition when it is cooled down to -60ºC, which is significantly lower than the homogeneous ice nucleation temperature [Bronshteyn, V.L. et al. Biophys. J. 65 (1993) 1853].

Mirror energy difference and the structure of loosely bound proton-rich nuclei around A =20

Science.gov (United States)

Yuan, Cenxi; Qi, Chong; Xu, Furong; Suzuki, Toshio; Otsuka, Takaharu

2014-04-01

The properties of loosely bound proton-rich nuclei around A =20 are investigated within the framework of the nuclear shell model. In these nuclei, the strength of the effective interactions involving the loosely bound proton s1/2 orbit is significantly reduced in comparison with that of those in their mirror nuclei. We evaluate the reduction of the effective interaction by calculating the monopole-based-universal interaction (VMU) in the Woods-Saxon basis. The shell-model Hamiltonian in the sd shell, such as USD, can thus be modified to reproduce the binding energies and energy levels of the weakly bound proton-rich nuclei around A =20. The effect of the reduction of the effective interaction on the structure and decay properties of these nuclei is also discussed.

Class I Cytokine Receptors: Structure and function in the Membrane

DEFF Research Database (Denmark)

Bugge, Katrine Østergaard

bilayer via structural characterizations of TMD representatives. To enable structural studies of these domains, an organic-extraction based strategy for efficient production of isotope-labeled TMDs with or without short intrinsically disordered regions was developed. This strategy successfully provided...... of these challenging domains. Supplemented by a review of the current collection of TMD structures from single-pass transmembrane receptors, the thesis as a whole provides important insights on the structure and function in the membrane as well as highlight the open questions to be addressed in the years to come.......Class I cytokine receptors are involved in important biological functions of both physiological and pathological nature in mammals. However, the molecular details of the cross-membrane signal transduction through these receptors remain obscure. One of the major reasons for this is the lack...

Structure of the [delta]-opioid receptor bound to naltrindole

Energy Technology Data Exchange (ETDEWEB)

Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K. (Stanford-MED)

2012-07-11

The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

Critical Structure for Telescopic Movement of Honey bee (Insecta: Apidae) Abdomen: Folded Intersegmental Membrane.

Science.gov (United States)

Zhao, Jieliang; Yan, Shaoze; Wu, Jianing

2016-01-01

The folded intersegmental membrane is a structure that interconnects two adjacent abdominal segments; this structure is distributed in the segments of the honey bee abdomen. The morphology of the folded intersegmental membrane has already been documented. However, the ultrastructure of the intersegmental membrane and its assistive role in the telescopic movements of the honey bee abdomen are poorly understood. To explore the morphology and ultrastructure of the folded intersegmental membrane in the honey bee abdomen, frozen sections were analyzed under a scanning electron microscope. The intersegmental membrane between two adjacent terga has a Z-S configuration that greatly influences the daily physical activities of the honey bee abdomen. The dorsal intersegmental membrane is 2 times thicker than the ventral one, leading to asymmetric abdominal motion. Honey bee abdominal movements were recorded using a high-speed camera and through phase-contrast computed tomography. These movements conformed to the structural features of the folded intersegmental membrane. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.

Dynamic analysis and reliability assessment of structures with uncertain-but-bounded parameters under stochastic process excitations

International Nuclear Information System (INIS)

Do, Duy Minh; Gao, Wei; Song, Chongmin; Tangaramvong, Sawekchai

2014-01-01

This paper presents the non-deterministic dynamic analysis and reliability assessment of structures with uncertain-but-bounded parameters under stochastic process excitations. Random ground acceleration from earthquake motion is adopted to illustrate the stochastic process force. The exact change ranges of natural frequencies, random vibration displacement and stress responses of structures are investigated under the interval analysis framework. Formulations for structural reliability are developed considering the safe boundary and structural random vibration responses as interval parameters. An improved particle swarm optimization algorithm, namely randomised lower sequence initialized high-order nonlinear particle swarm optimization algorithm, is employed to capture the better bounds of structural dynamic characteristics, random vibration responses and reliability. Three numerical examples are used to demonstrate the presented method for interval random vibration analysis and reliability assessment of structures. The accuracy of the results obtained by the presented method is verified by the randomised Quasi-Monte Carlo simulation method (QMCSM) and direct Monte Carlo simulation method (MCSM). - Highlights: • Interval uncertainty is introduced into structural random vibration responses. • Interval dynamic reliability assessments of structures are implemented. • Boundaries of structural dynamic response and reliability are achieved

Chloroplast protein synthesis: thylakoid bound polysomes synthesize thylakoid proteins

International Nuclear Information System (INIS)

Hurewitz, J.; Jagendorf, A.T.

1986-01-01

Previous work indicated more polysomes bound to pea thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus the major effect of light in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus translation initiation and termination probably control the cycling of bound ribosomes. While only 3 to 6% of total RNA is in bound polysomes the incorporation of 3 H-Leu into thylakoids was proportional to the amount of this bound RNA. When Micrococcal nuclease-treated thylakoids were added to labeled runoff translation products of stroma ribosomes, less than 1% of the label adhered to the added membranes; but 37% of the labeled products made by thylakoid polysomes were bound. These data support the concept that stroma ribosomes are recruited into thylakoid proteins

Membrane Transporters: Structure, Function and Targets for Drug Design

Science.gov (United States)

Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

Biomimetic membranes and methods of making biomimetic membranes

Science.gov (United States)

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

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

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Lomize Mikhail A

2007-06-01

Full Text Available Abstract 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

Pathogen-expanded CD11b+ invariant NKT cells feedback inhibit T cell proliferation via membrane-bound TGF-β1.

Science.gov (United States)

Han, Yanmei; Jiang, Zhengping; Chen, Zhubo; Gu, Yan; Liu, Yanfang; Zhang, Xiang; Cao, Xuetao

2015-04-01

Natural killer T cells (NKT cells) are effector cells, but also regulator of immune response, which either promote or suppress immune response through production of different cytokines. However, the subsets of NKT cells with definite phenotype and regulatory function need to be further identified. Furthermore, the mechanisms for NKT cells to regulate immune response remain to be fully elucidated. Here we identified CD11b(+) invariant NKT (CD11b(+) iNKT) cells as a new subset of regulatory NKT cells in mouse models with infection. αGalCer:CD1d complex(+)TCRβ(+)NK1.1(+) NKT cells could be categorized to CD11b(+) and CD11b(-) subsets. NKT cells are enriched in liver. During Listeria monocytogenes infection, hepatic CD11b(+) iNKT cells were significantly induced and expanded, with peak expansion on day 8. CD11b(+) iNKT cells were also expanded significantly in spleen and mesenteric lymph nodes. As compared to CD11b(-) iNKT cells, CD11b(+) iNKT cells expressed higher levels of CD27, FasL, B7H1, CD69, and particularly higher level of membrane-bound TGF-β1 (mTGF-β1), but produced less IFN-γ, IL-4, IL-10 and TGF-β1. Hepatic CD11b(+) iNKT cells suppressed antigen-nonspecific and OVA-specific CD4 and CD8 T cell proliferation through mTGF-β1 both in vitro and in vivo, meanwhile, they did not interfere with activation of CD4 T cells and cytotoxicity of the activated CD8 T cells. Thus, we have identified a new subset of pathogen-expanded CD11b(+) invariant NKT cells which can feedback inhibit T cell response through cell-to-cell contact via cell surface (membrane-bound) TGF-β1, especially at the late stage of immune response against infection. CD11b(+) regulatory iNKT cells may contribute to protect host from pathological injure by preventing immune overactivation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modification of track membranes structure by gas discharge etching method

International Nuclear Information System (INIS)

Dmitriev, S.N.; Kravets, L.I.

1996-01-01

An investigation of the properties of polyethyleneterephthalate track membranes (PET TM) treated with the plasma RF-discharge in air has been performed. The influence of the plasma treatment conditions on the basic properties of the membranes, namely pore size and pore shape, porosity and mechanical strength has been studied. It was arranged that the effect of air plasma on the PET TM results to etching a membrane's surface layer. The membranes' pore size and the form in this case change. It is shown that it is possible to change the structure of track membranes directly by the gas discharge etching method. Depending on the choice of discharge parameters, it is possible to make etching either in a part of the channel or along the whole length of the pore channels. In both cases the membranes with an asymmetric pore shape are formed which possess higher porosity and flow rate. The use of the membranes of such a type allows one to increase drastically the efficiency of the filtration processes. 12 refs., 5 figs., 1 tab

Data supporting beta-amyloid dimer structural transitions and protein–lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures

Directory of Open Access Journals (Sweden)

Sara Y. Cheng

2016-06-01

Full Text Available This data article supports the research article entitled “Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface” [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes.

Structure and interactions in biomaterials based on membrane-biopolymer self-assembly

Science.gov (United States)

Koltover, Ilya

Physical and chemical properties of artificial pure lipid membranes have been extensively studied during the last two decades and are relatively well understood. However, most real membrane systems of biological and biotechnological importance incorporate macromolecules either embedded into the membranes or absorbed onto their surfaces. We have investigated three classes of self-assembled membrane-biopolymer biomaterials: (i) Structure, interactions and stability of the two-dimensional crystals of the integral membrane protein bacteriorhodopsin (bR). We have conducted a synchrotron x-ray diffraction study of oriented bR multilayers. The important findings were as follows: (1) the protein 2D lattice exhibited diffraction patterns characteristic of a 2D solid with power-law decay of in-plane positional correlations, which allowed to measure the elastic constants of protein crystal; (2) The crystal melting temperature was a function of the multilayer hydration, reflecting the effect of inter-membrane repulsion on the stability of protein lattice; (3) Preparation of nearly perfect (mosaicity gene therapy applications. We have established that DNA complexes with cationic lipid (DOTAP) and a neutral lipid (DOPC) have a compact multilayer liquid crystalline structure ( L ca ) with DNA intercalated between the lipid bilayers in a periodic 2D smectic phase. Furthermore, a different 2D columnar phase of complexes was found in mixtures with a transfectionen-hancing lipid DOPE. This structure ( HcII ) derived from synchrotron x-ray diffraction consists of DNA coated by cationic lipid monolayers and arranged on a two-dimensional hexagonal lattice. Optical microscopy revealed that the L ca complexes bind stably to anionic vesicles (models of cellular membranes), whereas the more transfectant HcII complexes are unstable, rapidly fusing and releasing DNA upon adhering to anionic vesicles.

Crystal Structures of SlyA Protein, a Master Virulence Regulator of Salmonella, in Free and DNA-bound States

Energy Technology Data Exchange (ETDEWEB)

Dolan, Kyle T.; Duguid, Erica M.; He, Chuan (UC)

2011-11-17

SlyA is a master virulence regulator that controls the transcription of numerous genes in Salmonella enterica. We present here crystal structures of SlyA by itself and bound to a high-affinity DNA operator sequence in the slyA gene. SlyA interacts with DNA through direct recognition of a guanine base by Arg-65, as well as interactions between conserved Arg-86 and the minor groove and a large network of non-base-specific contacts with the sugar phosphate backbone. Our structures, together with an unpublished structure of SlyA bound to the small molecule effector salicylate (Protein Data Bank code 3DEU), reveal that, unlike many other MarR family proteins, SlyA dissociates from DNA without large conformational changes when bound to this effector. We propose that SlyA and other MarR global regulators rely more on indirect readout of DNA sequence to exert control over many genes, in contrast to proteins (such as OhrR) that recognize a single operator.

Liaison of 3H 5-HT and adenyl cyclasic activation induced by the 5-HT in preparations of brain glial membranes

International Nuclear Information System (INIS)

Fillion, Gilles; Beaudoin, Dominique; Rousselle, J.-C.; Jacob, Joseph

1980-01-01

Purified glial membrane preparations have been isolated from horse brain striatum. Tritiated 5-HT bound to these membranes with a high affinity (K(D)=10 nM); the corresponding bindings is reversible and appears specific of the serotoninergic structure. In parallel, 5-HT activates an adenylate cyclase with a low affinity (K(D)=1 μM). The sites involved in this binding and in this adenylate cyclase activation appear different from the serotoninergic sites reported in the neuronal membrane preparations [fr

Membrane-lipid therapy: A historical perspective of membrane-targeted therapies - From lipid bilayer structure to the pathophysiological regulation of cells.

Science.gov (United States)

Escribá, Pablo V

2017-09-01

Our current understanding of membrane lipid composition, structure and functions has led to the investigation of their role in cell signaling, both in healthy and pathological cells. As a consequence, therapies based on the regulation of membrane lipid composition and structure have been recently developed. This novel field, known as Membrane Lipid Therapy, is growing and evolving rapidly, providing treatments that are now in use or that are being studied for their application to oncological disorders, Alzheimer's disease, spinal cord injury, stroke, diabetes, obesity, and neuropathic pain. This field has arisen from relevant discoveries on the behavior of membranes in recent decades, and it paves the way to adopt new approaches in modern pharmacology and nutrition. This innovative area will promote further investigation into membranes and the development of new therapies with molecules that target the cell membrane. Due to the prominent roles of membranes in the cells' physiology and the paucity of therapeutic approaches based on the regulation of the lipids they contain, it is expected that membrane lipid therapy will provide new treatments for numerous pathologies. The first on-purpose rationally designed molecule in this field, minerval, is currently being tested in clinical trials and it is expected to enter the market around 2020. However, it seems feasible that during the next few decades other membrane regulators will also be marketed for the treatment of human pathologies. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017. Published by Elsevier B.V.

Reconciliation of opposing views on membrane-sugar interactions

DEFF Research Database (Denmark)

Andersen, Heidi D.; Wang, Chunhua; Arleth, Lise

2011-01-01

It is well established that small sugars exert different types of stabilization of biomembranes both in vivo and in vitro. However, the essential question of whether sugars are bound to or expelled from membrane surfaces, i.e., the sign and size of the free energy of the interaction, remains...... unresolved, and this prevents a molecular understanding of the stabilizing mechanism. We have used small-angle neutron scattering and thermodynamic measurements to show that sugars may be either bound or expelled depending on the concentration of sugar. At low concentration, small sugars bind quite strongly...... to a lipid bilayer, and the accumulation of sugar at the interface makes the membrane thinner and laterally expanded. Above â¼0.2 M the sugars gradually become expelled from the membrane surface, and this repulsive mode of interaction counteracts membrane thinning. The dual nature of sugar...

The structure of the COPII transport-vesicle coat assembled on membranes.

Science.gov (United States)

Zanetti, Giulia; Prinz, Simone; Daum, Sebastian; Meister, Annette; Schekman, Randy; Bacia, Kirsten; Briggs, John A G

2013-09-17

Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:http://dx.doi.org/10.7554/eLife.00951.001.

The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

Directory of Open Access Journals (Sweden)

Helene J Bustad

Full Text Available Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

Detection of membrane-bound and soluble antigens by magnetic levitation

DEFF Research Database (Denmark)

Andersen, Mikkel Schou; Howard, Emily; Lu, Shulin

2017-01-01

blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its...

Structural characterization of coatomer in its cytosolic state

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Shengliu Wang

2016-07-01

Full Text Available Abstract Studies on coat protein I (COPI have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

Environmental behaviour of tensile membrane structures

OpenAIRE

Elnokaly, Amira; Chilton, John; Wilson, Robin

2002-01-01

This paper considers the environmental properties of spaces enclosed by tensile membrane structures (TMS). Limitations in the understanding of the environmental and thermal performance of TMS have to some extent hindered their acceptance by building clients and the building industry. A review of the early attempts to model the thermal environment of spaces enclosed by TMS is given and their environmental and thermal properties are discussed. The lack of appropriate tools for the investigation...

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles.

Science.gov (United States)

Schibli, D J; Hwang, P M; Vogel, H J

1999-03-12

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.

An investigation on polymeric blend mixed matrix membranes of ...

African Journals Online (AJOL)

Polymeric membranes have been vastly used for gas separation purposes however they have an upper-bound trade off problem which is the reason why this research work is focusing on inorganic filler added to polymer blend membranes to enhance the selectivity and permeability of the resulted membranes. Different ...

An experimental study of perovskite-structured mixed ionic- electronic conducting oxides and membranes

Science.gov (United States)

Zeng, Pingying

In recent decades, ceramic membranes based on mixed ionic and electronic conducting (MIEC) perovskite-structured oxides have received many attentions for their applications for air separation, or as a membrane reactor for methane oxidation. While numerous perovskite oxide materials have been explored over the past two decades; there are hardly any materials with sufficient practical economic value and performance for large scale applications, which justifies continuing the search for new materials. The main purposes of this thesis study are: (1) develop several novel SrCoO3-delta based MIEC oxides, SrCoCo1-xMxO3-delta, based on which membranes exhibit excellent oxygen permeability; (2) investigate the significant effects of the species and concentration of the dopants M (metal ions with fixed valences) on the various properties of these membranes; (3) investigate the significant effects of sintering temperature on the microstructures and performance of oxygen permeation membranes; and (4) study the performance of oxygen permeation membranes as a membrane reactor for methane combustion. To stabilize the cubic phase structure of the SrCoO3-delta oxide, various amounts of scandium was doped into the B-site of SrCoO 3-delta to form a series of new perovskite oxides, SrScxCoCo 1-xO3-delta (SSCx, x = 0-0.7). The significant effects of scandium-doping concentration on the phase structure, electrical conductivity, sintering performance, thermal and structural stability, cathode performance, and oxygen permeation performance of the SSCx membranes, were systematically studied. Also for a more in-depth understanding, the rate determination steps for the oxygen transport process through the membranes were clarified by theoretical and experimental investigation. It was found that only a minor amount of scandium (5 mol%) doping into the B-site of SrCoO3-delta can effectively stabilize the cubic phase structure, and thus significantly improve the electrical conductivity and

Understanding the structure and performance of self-assembled triblock terpolymer membranes

KAUST Repository

Pendergast, MaryTheresa M.; Mika Dorin, Rachel; Phillip, William A.; Wiesner, Ulrich; Hoek, Eric M.V.

2013-01-01

Nanoporous membranes represent a possible route towards more precise particle and macromolecular separations, which are of interest across many industries. Here, we explored membranes with vertically-aligned nanopores formed from a poly(isoprene-. b-styrene-. b-4 vinyl pyridine) (ISV) triblock terpolymer via a hybrid self-assembly/nonsolvent induced phase separation process (S-NIPS). ISV concentration, solvent composition, and evaporation time in the S-NIPS process were varied to tailor ordering of the selective layer and produce enhanced water permeability. Here, water permeability was doubled over previous versions of ISV membranes. This was achieved by increasing volatile solvent concentration, thereby decreasing the evaporation period required for self-assembly. Fine-tuning was required, however, since overly-rapid evaporation did not yield the desired pore structure. Transport models, used to relate the in-. situ structure to the performance of these materials, revealed narrowing of pores and blocking by the dense region below. It was shown that these vertically aligned nanoporous membranes compare favorably with commercial ultrafiltration membranes formed by NIPS and track-etching processes, which suggests that there is practical value in further developing and optimizing these materials for specific industrial separations. © 2013 Elsevier B.V.

BCL::MP-Fold: membrane protein structure prediction guided by EPR restraints

Science.gov (United States)

Fischer, Axel W.; Alexander, Nathan S.; Woetzel, Nils; Karakaş, Mert; Weiner, Brian E.; Meiler, Jens

2016-01-01

For many membrane proteins, the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The protein-size-normalized root-mean-square-deviation (RMSD100) value of the most accurate model is better than 8 Å for twenty-seven, better than 6 Å for twenty-two, and better than 4 Å for fifteen out of twenty-nine proteins, demonstrating the algorithm’s ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data. PMID:25820805

Understanding the structure and performance of self-assembled triblock terpolymer membranes

KAUST Repository

Pendergast, MaryTheresa M.

2013-10-01

Nanoporous membranes represent a possible route towards more precise particle and macromolecular separations, which are of interest across many industries. Here, we explored membranes with vertically-aligned nanopores formed from a poly(isoprene-. b-styrene-. b-4 vinyl pyridine) (ISV) triblock terpolymer via a hybrid self-assembly/nonsolvent induced phase separation process (S-NIPS). ISV concentration, solvent composition, and evaporation time in the S-NIPS process were varied to tailor ordering of the selective layer and produce enhanced water permeability. Here, water permeability was doubled over previous versions of ISV membranes. This was achieved by increasing volatile solvent concentration, thereby decreasing the evaporation period required for self-assembly. Fine-tuning was required, however, since overly-rapid evaporation did not yield the desired pore structure. Transport models, used to relate the in-. situ structure to the performance of these materials, revealed narrowing of pores and blocking by the dense region below. It was shown that these vertically aligned nanoporous membranes compare favorably with commercial ultrafiltration membranes formed by NIPS and track-etching processes, which suggests that there is practical value in further developing and optimizing these materials for specific industrial separations. © 2013 Elsevier B.V.

Mechanism of Shiga Toxin Clustering on Membranes

DEFF Research Database (Denmark)

Pezeshkian, Weria; Gao, Haifei; Arumugam, Senthil

2017-01-01

between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga...... toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface...... molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications....

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

Science.gov (United States)

Bansal, Sunil; Durrett, Timothy P.

2016-01-01

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

Uniaxial and biaxial tensioning effects on thin membrane materials. [large space structures

Science.gov (United States)

Hinson, W. F.; Goslee, J. W.

1980-01-01

Thin laminated membranes are being considered for various surface applications on future large space structural systems. Some of the thin membranes would be stretched across or between structural members with the requirement that the membrane be maintained within specified limits of smoothness which would be dictated by the particular applications such as antenna reflector requirements. The multiaxial tensile force required to maintain the smoothness in the membrane needs to be determined for use in the structure design. Therefore, several types of thicknesses of thin membrane materials have been subjected to varied levels of uniaxial and biaxial tensile loads. During the biaxial tests, deviations of the material surface smoothness were measured by a noncontacting capacitance probe. Basic materials consisted of composites of vacuum deposited aluminum on Mylar and Kapton ranging in thickness from 0.00025 in (0.000635 cm) to 0.002 in (0.00508 cm). Some of the material was reinforced with Kevlar and Nomex scrim. The uniaxial tests determined the material elongation and tensile forces up to ultimate conditions. Biaxial tests indicated that a relatively smooth material surface could be achieved with tensile force of approximately 1 to 15 Newtons per centimeter, depending upon the material thickness and/or reinforcement.

Partially Fluorinated Sulfonated Poly(ether amide Fuel Cell Membranes: Influence of Chemical Structure on Membrane Properties

Directory of Open Access Journals (Sweden)

Chulsung Bae

2011-01-01

Full Text Available A series of fluorinated sulfonated poly (ether amides (SPAs were synthesized for proton exchange membrane fuel cell applications. A polycondensation reaction of 4,4’-oxydianiline, 2-sulfoterephthalic acid monosodium salt, and tetrafluorophenylene dicarboxylic acids (terephthalic and isophthalic or fluoroaliphatic dicarboxylic acids produced SPAs with sulfonation degrees of 80–90%. Controlling the feed ratio of the sulfonated and unsulfonated dicarboxylic acid monomers afforded random SPAs with ion exchange capacities between 1.7 and 2.2 meq/g and good solubility in polar aprotic solvents. Their structures were characterized using NMR and FT IR spectroscopies. Tough, flexible, and transparent films were obtained with dimethylsulfoxide using a solution casting method. Most SPA membranes with 90% sulfonation degree showed high proton conductivity (>100 mS/cm at 80 °C and 100% relative humidity. Among them, two outstanding ionomers (ODA-STA-TPA-90 and ODA-STA-IPA-90 showed proton conductivity comparable to that of Nafion 117 between 40 and 80 °C. The influence of chemical structure on the membrane properties was systematically investigated by comparing the fluorinated polymers to their hydrogenated counterparts. The results suggest that the incorporation of fluorinated moieties in the polymer backbone of the membrane reduces water absorption. High molecular weight and the resulting physical entanglement of the polymers chains played a more important role in improving stability in water, however.

Relating performance of thin-film composite forward osmosis membranes to support layer formation and structure

KAUST Repository

Tiraferri, Alberto

2011-02-01

Osmotically driven membrane processes have the potential to treat impaired water sources, desalinate sea/brackish waters, and sustainably produce energy. The development of a membrane tailored for these processes is essential to advance the technology to the point that it is commercially viable. Here, a systematic investigation of the influence of thin-film composite membrane support layer structure on forward osmosis performance is conducted. The membranes consist of a selective polyamide active layer formed by interfacial polymerization on top of a polysulfone support layer fabricated by phase separation. By systematically varying the conditions used during the casting of the polysulfone layer, an array of support layers with differing structures was produced. The role that solvent quality, dope polymer concentration, fabric layer wetting, and casting blade gate height play in the support layer structure formation was investigated. Using a 1M NaCl draw solution and a deionized water feed, water fluxes ranging from 4 to 25Lm-2h-1 with consistently high salt rejection (>95.5%) were produced. The relationship between membrane structure and performance was analyzed. This study confirms the hypothesis that the optimal forward osmosis membrane consists of a mixed-structure support layer, where a thin sponge-like layer sits on top of highly porous macrovoids. Both the active layer transport properties and the support layer structural characteristics need to be optimized in order to fabricate a high performance forward osmosis membrane. © 2010 Elsevier B.V.

Structural Study and Modification of Support Layer for Forward Osmosis Membranes

KAUST Repository

Shi, Meixia

2016-06-01

Water scarcity is a serious global issue, due to the increasing population and developing economy, and membrane technology is an essential way to address this problem. Forward osmosis (FO) is an emerging membrane process, due to its low energy consumption (not considering the draw solute regeneration). A bottleneck to advance this technology is the design of the support layer for FO membranes to minimize the internal concentration polarization. In this dissertation, we focus on the structural study and modification of the support layer for FO membranes. Firstly, we digitally reconstruct different membrane morphologies in 3D and propose a method for predicting performance in ultrafiltration operations. Membranes with analogous morphologies are later used as substrate for FO membranes. Secondly, we experimentally apply substrates with different potentially suitable morphologies as an FO support layer. We investigate their FO performance after generating a selective polyamide layer on the top, by interfacial polymerization. Among the different substrates we include standard asymmetric porous membranes prepared from homopolymers, such as polysulfone. Additionally block copolymer membrane and Anodisc alumina membrane are chosen based on their exceptional structures, with cylindrical pores at least in part. 3D digitally reconstructed porous substrates, analogous to those investigated for ultrafiltration, are then used to model the performance in FO operation. Finally, we analyze the effect of intermediate layers between the porous substrate and the interfacial polymerized layer. We investigate two materials including chitosan and hydrogel. The main results are the following. Pore-scale modeling for digital membrane generation effectively predicts the velocity profile in different layers of the membrane and the performance in UF experiments. Flow simulations confirm the advantage of finger-like substrates over sponge-like ones, when high water permeance is sought

Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system.

Science.gov (United States)

Takahara, Michiyo; Sakaue, Haruka; Onishi, Yukiko; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

2013-01-11

Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors. Copyright © 2012. Published by Elsevier Inc.

Cell-bound lipases from Burkholderia sp. ZYB002: gene sequence analysis, expression, enzymatic characterization, and 3D structural model.

Science.gov (United States)

Shu, Zhengyu; Lin, Hong; Shi, Shaolei; Mu, Xiangduo; Liu, Yanru; Huang, Jianzhong

2016-05-03

The whole-cell lipase from Burkholderia cepacia has been used as a biocatalyst in organic synthesis. However, there is no report in the literature on the component or the gene sequence of the cell-bound lipase from this species. Qualitative analysis of the cell-bound lipase would help to illuminate the regulation mechanism of gene expression and further improve the yield of the cell-bound lipase by gene engineering. Three predictive cell-bound lipases, lipA, lipC21 and lipC24, from Burkholderia sp. ZYB002 were cloned and expressed in E. coli. Both LipA and LipC24 displayed the lipase activity. LipC24 was a novel mesophilic enzyme and displayed preference for medium-chain-length acyl groups (C10-C14). The 3D structural model of LipC24 revealed the open Y-type active site. LipA displayed 96 % amino acid sequence identity with the known extracellular lipase. lipA-inactivation and lipC24-inactivation decreased the total cell-bound lipase activity of Burkholderia sp. ZYB002 by 42 % and 14 %, respectively. The cell-bound lipase activity from Burkholderia sp. ZYB002 originated from a multi-enzyme mixture with LipA as the main component. LipC24 was a novel lipase and displayed different enzymatic characteristics and structural model with LipA. Besides LipA and LipC24, other type of the cell-bound lipases (or esterases) should exist.

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

International Nuclear Information System (INIS)

Miles, Andrew J.; Fedosova, Natalya U.; Hoffmann, Søren V.; Wallace, B.A.; Esmann, Mikael

2013-01-01

Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

Energy Technology Data Exchange (ETDEWEB)

Miles, Andrew J. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Fedosova, Natalya U. [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark); Hoffmann, Søren V. [ISA, Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus (Denmark); Wallace, B.A. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Esmann, Mikael, E-mail: me@biophys.au.dk [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark)

2013-05-31

Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

Identification and Structural Characterization of Novel A-kinase Anchoring Proteins

NARCIS (Netherlands)

Burgers, P.P.

2014-01-01

The work described in this thesis initially focusses on the discovery of a novel PKA-RI specific AKAP: small membrane AKAP (smAKAP). Afterwards we centre on the structural interaction between smAKAP and PKA-RI to reveal the first PKA-RI specific AKAP bound to PKA-RI crystal structure. Interestingly,

Different Structures of PVA Nanofibrous Membrane for Sound Absorption Application

Directory of Open Access Journals (Sweden)

Jana Mohrova

2012-01-01

Full Text Available The thin nanofibrous layer has different properties in the field of sound absorption in comparison with porous fibrous material which works on a principle of friction of air particles in contact with walls of pores. In case of the thin nanofibrous layer, which represents a sound absorber here, the energy of sonic waves is absorbed by the principle of membrane resonance. The structure of the membrane can play an important role in the process of converting the sonic energy to a different energy type. The vibration system acts differently depending on the presence of smooth fibers in the structure, amount of partly merged fibers, or structure of polymer foil as extreme. Polyvinyl alcohol (PVA was used as a polymer because of its good water solubility. It is possible to influence the structure of nanofibrous layer during the production process thanks to this property of polyvinyl alcohol.

Kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

Energy Technology Data Exchange (ETDEWEB)

Peters, R.L.; van Kooten, O.; Vredenberg, W.J.

1985-08-01

The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash-induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10(-8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.

Plant membranes a biophysical approach to structure, development and senescence

CERN Document Server

Leshem, Ya’Acov Y

1992-01-01

The plasma membrane is at once the window through which the cell senses the environment and the portal through which the environment influences the structure and activities of the cell. Its importance in cellular physiology can thus hardly be overestimated, since constant flow of materials between cell and environment is essential to the well-being of any biological system. The nature of the materials mov­ ing into the cell is also critical, since some substances are required for maintenance and growth, while others, because of their toxicity, must either be rigorously excluded or permitted to enter only after chemical alteration. Such alteration frequently permits the compounds to be sequestered in special cellular compartments having different types of membranes. This type of homogeneity, plus the fact that the wear and tear of transmembrane molecular traffic compels the system to be constantly monitored and repaired, means that the membrane system of any organism must be both structurally complex and dy­...

Characterization and quantitation of concanavalin A binding by plasma membrane enriched fractions from soybean root

International Nuclear Information System (INIS)

Berkowitz, R.L.; Travis, R.L.

1981-01-01

The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritated ( 3 H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of 3 H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10 15 molecules of 3 H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of α-methyl mannoside. A major peak of 3 H-Con A binding was also observed in fractions recovered from the 25/30% interface of a complex discontinuous sucrose density gradient when membranes were isolated in the absence of Mg 2+ . When high Mg 2+ was present in the isolation and gradient media, the peak was shifted to a fraction recovered from the 34/38% sucrose interface. These results suggest that Con A binding sites are also present on membranes of the endoplasmic reticulum. The amount of Con A bound by endoplasmic reticulum membranes was at least twice the amount bound by membranes in plasma membrane enriched fractions when binding was compared on a per unit membrane protein basis. In contrast, mitochondrial inner membranes, which equilibrate at the same density as plasma membranes, had little ability to bind the lectin

Dynamical and structural properties of lipid membranes in relation to liposomal drug delivery systems

DEFF Research Database (Denmark)

Jørgensen, Kent; Høyrup, Lise Pernille Kristine; Pedersen, Tina B.

2001-01-01

The structural and dynamical properties of DPPC liposomes containing lipopolymers (PEG-lipids) and charged DPPS lipids have been,studied in relation to the lipid membrane interaction of enzymes and peptides. The results suggest that both the lipid membrane structure and dynamics and in particular...

Detergent-dependent separation of postsynaptic density, membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain.

Science.gov (United States)

Zhao, LiYing; Sakagami, Hiroyuki; Suzuki, Tatsuo

2014-10-01

We systematically investigated the purification process of post-synaptic density (PSD) and post-synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX-100, n-octyl β-d-glucoside (OG) or 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX-100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD-PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX-100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft-associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level. We systematically investigated the purification process of post-synaptic density (PSD) and synaptic membrane rafts by examining the structures obtained after treatment of the SPMs with TX-100, n-octyl β-d-glucoside or CHAPSO. Differential distribution of type I and type II PSD, synaptic membrane rafts, and other novel subdomains in the SPM give clues to understand the structural organization of synapses at the molecular level. © 2014 International Society for Neurochemistry.

Drug binding and mobility relating to the thermal fluctuation in fluid lipid membranes

Science.gov (United States)

Okamura, Emiko; Yoshii, Noriyuki

2008-12-01

Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient F19 and H1 NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is ˜0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 1. Product-Bound Structures

Energy Technology Data Exchange (ETDEWEB)

Liu, Dali; Momb, Jessica; Thomas, Pei W.; Moulin, Aaron; Petsko, Gregory A.; Fast, Walter; Ringe, Dagmar (Brandeis); (Texas)

2008-08-06

Enzymes capable of hydrolyzing N-acyl-l-homoserine lactones (AHLs) used in some bacterial quorum-sensing pathways are of considerable interest for their ability to block undesirable phenotypes. Most known AHL hydrolases that catalyze ring opening (AHL lactonases) are members of the metallo-{beta}-lactamase enzyme superfamily and rely on a dinuclear zinc site for catalysis and stability. Here we report the three-dimensional structures of three product complexes formed with the AHL lactonase from Bacillus thuringiensis. Structures of the lactonase bound with two different concentrations of the ring-opened product of N-hexanoyl-l-homoserine lactone are determined at 0.95 and 1.4 {angstrom} resolution and exhibit different product configurations. A structure of the ring-opened product of the non-natural N-hexanoyl-l-homocysteine thiolactone at 1.3 {angstrom} resolution is also determined. On the basis of these product-bound structures, a substrate-binding model is presented that differs from previous proposals. Additionally, the proximity of the product to active-site residues and observed changes in protein conformation and metal coordination provide insight into the catalytic mechanism of this quorum-quenching metalloenzyme.

Bounds on isocurvature perturbations from cosmic microwave background and large scale structure data.

Science.gov (United States)

Crotty, Patrick; García-Bellido, Juan; Lesgourgues, Julien; Riazuelo, Alain

2003-10-24

We obtain very stringent bounds on the possible cold dark matter, baryon, and neutrino isocurvature contributions to the primordial fluctuations in the Universe, using recent cosmic microwave background and large scale structure data. Neglecting the possible effects of spatial curvature, tensor perturbations, and reionization, we perform a Bayesian likelihood analysis with nine free parameters, and find that the amplitude of the isocurvature component cannot be larger than about 31% for the cold dark matter mode, 91% for the baryon mode, 76% for the neutrino density mode, and 60% for the neutrino velocity mode, at 2sigma, for uncorrelated models. For correlated adiabatic and isocurvature components, the fraction could be slightly larger. However, the cross-correlation coefficient is strongly constrained, and maximally correlated/anticorrelated models are disfavored. This puts strong bounds on the curvaton model.

Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

Science.gov (United States)

Wang, Yukun; Chen, Charles H.; Hu, Dan; Ulmschneider, Martin B.; Ulmschneider, Jakob P.

2016-11-01

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures--formed by a single sequence--may be a key feature in preventing bacterial resistance and could explain why sequence-function relationships in AMPs remain elusive.

High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules

Science.gov (United States)

Baker, Bo Y; Shi, Wuxian; Wang, Benlian; Palczewski, Krzysztof

2014-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4, and bGAPDH(NAD)3. The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54–56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies. PMID:25176140

Wall-based identification of coherent structures in wall-bounded turbulence

Science.gov (United States)

Sanmiguel Vila, C.; Flores, O.

2018-04-01

During the last decades, a number of reduced order models based on coherent structures have been proposed to describe wall-bounded turbulence. Many of these models emphasize the importance of coherent wall-normal velocity eddies (ν-eddies), which drive the generation of the very long streamwise velocity structures observed in the logarithmic and outer region. In order to use these models to improve our ability to control wall-bounded turbulence in realistic applications, these ν-eddies need to be identified from the wall in a non-intrusive way. In this paper, the possibility of using the pressure signal at the wall to identify these ν-eddies is explored, analyzing the cross-correlation between the wall-normal velocity component and the pressure fluctuations at the wall in a DNS of a turbulent channel flow at Reτ = 939. The results show that the cross-correlation has a region of negative correlation upstream, and a region of positive correlation backwards. In the spanwise direction the correlation decays monotonously, except very close to the wall where a change of sign of the correlation coefficient is observed. Moreover, filtering the pressure fluctuations at the wall in space results in an increase of the region where the cross-correlation is strong, both for the positively and the negatively correlated regions. The use of a time filter for the pressure fluctuations at the wall yields different results, displacing the regions of strong correlation without changing much their sizes. The results suggest that space-filtering the pressure at the wall is a feasible way to identify ν-eddies of different sizes, which could be used to trigger turbulent control strategies.

Crystal Structure of the Oxazolidinone Antibiotic Linezolid Bound to the 50S Ribosomal Subunit

Energy Technology Data Exchange (ETDEWEB)

Ippolito,J.; Kanyo, Z.; Wang, D.; Franceschi, F.; Moore, P.; Steitz, T.; Duffy, E.

2008-01-01

The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

Membrane-bound IL-12 and IL-23 serve as potent mucosal adjuvants when co-presented on whole inactivated influenza vaccines.

Science.gov (United States)

Khan, Tila; Heffron, Connie L; High, Kevin P; Roberts, Paul C

2014-05-03

Potent and safe adjuvants are needed to improve the efficacy of parenteral and mucosal vaccines. Cytokines, chemokines and growth factors have all proven to be effective immunomodulatory adjuvants when administered with a variety of antigens. We have previously evaluated the efficacy of membrane-anchored interleukins (IL) such as IL-2 and IL-4 co-presented as Cytokine-bearing Influenza Vaccines (CYT-IVACs) using a mouse model of influenza challenge. Here, we describe studies evaluating the parenteral and mucosal adjuvanticity of membrane-bound IL-12 and IL-23 CYT-IVACs in young adult mice. Mucosal immunization using IL-12 and IL-23 bearing whole influenza virus vaccine (WIV) was more effective at eliciting virus-specific nasal IgA and reducing viral lung burden following challenge compared to control WIV vaccinated animals. Both IL-12 and IL-23 bearing WIV elicited the highest anti-viral IgA levels in serum and nasal washes. This study highlights for the first time the mucosal adjuvant potential of IL-12 and IL-23 CYT-IVAC formulations in eliciting mucosal immune responses and reducing viral lung burden. The co-presentation of immunomodulators in direct context with viral antigen in whole inactivated viral vaccines may provide a means to significantly lower the dose of vaccine required for protection.

Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins.

Science.gov (United States)

Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij

2012-03-01

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/. Copyright © 2011 Wiley Periodicals, Inc.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies.

Science.gov (United States)

Zhang, Rongfu; Sahu, Indra D; Liu, Lishan; Osatuke, Anna; Comer, Raven G; Dabney-Smith, Carole; Lorigan, Gary A

2015-01-01

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment

Computational Approaches for Revealing the Structure of Membrane Transporters: Case Study on Bilitranslocase

Directory of Open Access Journals (Sweden)

Katja Venko

Full Text Available The structural and functional details of transmembrane proteins are vastly underexplored, mostly due to experimental difficulties regarding their solubility and stability. Currently, the majority of transmembrane protein structures are still unknown and this present a huge experimental and computational challenge. Nowadays, thanks to X-ray crystallography or NMR spectroscopy over 3000 structures of membrane proteins have been solved, among them only a few hundred unique ones. Due to the vast biological and pharmaceutical interest in the elucidation of the structure and the functional mechanisms of transmembrane proteins, several computational methods have been developed to overcome the experimental gap. If combined with experimental data the computational information enables rapid, low cost and successful predictions of the molecular structure of unsolved proteins. The reliability of the predictions depends on the availability and accuracy of experimental data associated with structural information. In this review, the following methods are proposed for in silico structure elucidation: sequence-dependent predictions of transmembrane regions, predictions of transmembrane helix–helix interactions, helix arrangements in membrane models, and testing their stability with molecular dynamics simulations. We also demonstrate the usage of the computational methods listed above by proposing a model for the molecular structure of the transmembrane protein bilitranslocase. Bilitranslocase is bilirubin membrane transporter, which shares similar tissue distribution and functional properties with some of the members of the Organic Anion Transporter family and is the only member classified in the Bilirubin Transporter Family. Regarding its unique properties, bilitranslocase is a potentially interesting drug target. Keywords: Membrane proteins, Bilitranslocase, 3D protein structure, Transmembrane region predictors, Helix–helix interactions

Self-ordered, controlled structure nanoporous membranes using constant current anodization.

Science.gov (United States)

Lee, Kwan; Tang, Yun; Ouyang, Min

2008-12-01

We report a constant current (CC) based anodization technique to fabricate and control structure of mechanically stable anodic aluminum oxide (AAO) membranes with a long-range ordered hexagonal nanopore pattern. For the first time we show that interpore distance (Dint) of a self-ordered nanopore feature can be continuously tuned over a broad range with CC anodization and is uniquely defined by the conductivity of sulfuric acid as electrolyte. We further demonstrate that this technique can offer new degrees of freedom for engineering planar nanopore structures by fine tailoring the CC based anodization process. Our results not only facilitate further understanding of self-ordering mechanism of alumina membranes but also provide a fast, simple (without requirement of prepatterning or preoxide layer), and flexible methodology for controlling complex nanoporous structures, thus offering promising practical applications in nanotechnology.

Bound nucleon structure function in the picture of relativistic constituent quarks

International Nuclear Information System (INIS)

Grigoryan, L.A.; Shakhbazyan, V.A.

1987-01-01

The structure function F 2N of nucleons in the deuterium, carbon and iron nuclei is calculated as a function of Q 2 in two approaches: taking into account the nucleon swelling in nuclei due to the partial deconfinement of quarks in nuclear medium; in the conventional approach of nuclear physics, taking into account the getting off the mass shell of the bound nucleon and Fermi motion in nucleons. It is shown that the conventional approach of nuclear physics does not explain the EMC effect in the region of small x

Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

International Nuclear Information System (INIS)

Wang, Jimin; Li, Yue; Modis, Yorgo

2014-01-01

The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed

Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

Energy Technology Data Exchange (ETDEWEB)

Wang, Jimin, E-mail: jimin.wang@yale.edu; Li, Yue; Modis, Yorgo, E-mail: yorgo.modis@yale.edu

2014-04-15

The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

Influence of cholesterol and ceramide VI on the structure of multilamellar lipid membranes at water exchange

International Nuclear Information System (INIS)

Ryabova, N. Yu.; Kiselev, M. A.; Balagurov, A. M.

2010-01-01

The structural changes in the multilamellar lipid membranes of dipalmitoylphosphatidylcholine (DPPC)/cholesterol and DPPC/ceramide VI binary systems during hydration and dehydration have been studied by neutron diffraction. The effect of cholesterol and ceramide on the kinetics of water exchange in DPPC membranes is characterized. Compared to pure DPPC, membranes of binary systems swell faster during hydration (with a characteristic time of ∼30 min). Both compounds, ceramide VI and cholesterol, similarly affect the hydration of DPPC membranes, increasing the repeat distance due to the bilayer growth. However, in contrast to cholesterol, ceramide significantly reduces the thickness of the membrane water layer. The introduction of cholesterol into a DPPC membrane slows down the change in the parameters of the bilayer internal structure during dehydration. In the DPPC/ceramide VI/cholesterol ternary system (with a molar cholesterol concentration of 40%), cholesterol is partially released from the lamellar membrane structure into the crystalline phase.

Detection of proteins on blot transfer membranes.

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Sasse, Joachim; Gallagher, Sean R

2003-11-01

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Hydro-osmotic Instabilities in Active Membrane Tubes

Science.gov (United States)

Al-Izzi, Sami C.; Rowlands, George; Sens, Pierre; Turner, Matthew S.

2018-03-01

We study a membrane tube with unidirectional ion pumps driving an osmotic pressure difference. A pressure-driven peristaltic instability is identified, qualitatively distinct from similar tension-driven Rayleigh-type instabilities on membrane tubes. We discuss how this instability could be related to the function and biogenesis of membrane bound organelles, in particular, the contractile vacuole complex. The unusually long natural wavelength of this instability is in agreement with that observed in cells.

Uniform Recovery Bounds for Structured Random Matrices in Corrupted Compressed Sensing

Science.gov (United States)

Zhang, Peng; Gan, Lu; Ling, Cong; Sun, Sumei

2018-04-01

We study the problem of recovering an $s$-sparse signal $\\mathbf{x}^{\\star}\\in\\mathbb{C}^n$ from corrupted measurements $\\mathbf{y} = \\mathbf{A}\\mathbf{x}^{\\star}+\\mathbf{z}^{\\star}+\\mathbf{w}$, where $\\mathbf{z}^{\\star}\\in\\mathbb{C}^m$ is a $k$-sparse corruption vector whose nonzero entries may be arbitrarily large and $\\mathbf{w}\\in\\mathbb{C}^m$ is a dense noise with bounded energy. The aim is to exactly and stably recover the sparse signal with tractable optimization programs. In this paper, we prove the uniform recovery guarantee of this problem for two classes of structured sensing matrices. The first class can be expressed as the product of a unit-norm tight frame (UTF), a random diagonal matrix and a bounded columnwise orthonormal matrix (e.g., partial random circulant matrix). When the UTF is bounded (i.e. $\\mu(\\mathbf{U})\\sim1/\\sqrt{m}$), we prove that with high probability, one can recover an $s$-sparse signal exactly and stably by $l_1$ minimization programs even if the measurements are corrupted by a sparse vector, provided $m = \\mathcal{O}(s \\log^2 s \\log^2 n)$ and the sparsity level $k$ of the corruption is a constant fraction of the total number of measurements. The second class considers randomly sub-sampled orthogonal matrix (e.g., random Fourier matrix). We prove the uniform recovery guarantee provided that the corruption is sparse on certain sparsifying domain. Numerous simulation results are also presented to verify and complement the theoretical results.

Bound states in string nets

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Schulz, Marc Daniel; Dusuel, Sébastien; Vidal, Julien

2016-11-01

We discuss the emergence of bound states in the low-energy spectrum of the string-net Hamiltonian in the presence of a string tension. In the ladder geometry, we show that a single bound state arises either for a finite tension or in the zero-tension limit depending on the theory considered. In the latter case, we perturbatively compute the binding energy as a function of the total quantum dimension. We also address this issue in the honeycomb lattice where the number of bound states in the topological phase depends on the total quantum dimension. Finally, the internal structure of these bound states is analyzed in the zero-tension limit.

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

Science.gov (United States)

Lai, Alex L.; Tamm, Lukas K.

2010-01-01

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

Fiber Temperature Sensor Based on Micro-mechanical Membranes and Optical Interference Structure

International Nuclear Information System (INIS)

Liu Yueming; Tian Weijian; Hua Jing

2011-01-01

A novel fiber temperature sensor is presented theoretically and experimentally in this paper. Its working principle is based on Optical Fabry-Perot interference structure that is formed between a polished optical fiber end and micro-mechanical Bi-layered membranes. When ambient temperature is varying, Bi-layered membranes will be deflected and the length of Fabry-Perot cavity will be changed correspondingly. By detecting the reflecting optical intensity from the Fabry-Perot cavity, the ambient temperature can be measured. Using finite element software ANSYS, the sensor structure was optimized based on optical Interference theory and Bi-layered membranes thermal expansion theory, and theoretical characteristics was simulated by computer software. In the end, using optical fiber 2x2 coupler and photo-electrical detector, the fabricated sample sensor was tested successfully by experiment that demonstrating above theoretical analysis and simulation results. This sensor has some favorable features, such as: micro size owing to its micro-mechanical structure, high sensitivity owing to its working Fabry-Perot interference cavity structure, and optical integration character by using optical fiber techniques.

Study of structural stability and damaging effect on membrane for four Aβ42 dimers.

Directory of Open Access Journals (Sweden)

Wei Feng

Full Text Available Increasing evidence shows that Aβ oligomers are key pathogenic molecules in Alzheimer's disease. Among Aβ oligomers, dimer is the smallest aggregate and toxic unit. Therefore, understanding its structural and dynamic properties is quite useful to prevent the formation and toxicity of the Aβ oligomers. In this study, we performed molecular dynamic simulations on four Aβ42 dimers, 2NCb, CNNC, NCNC and NCCN, within the hydrated DPPC membrane. Four Aβ42 dimers differ in the arrangements of two Aβ42 peptides. This study aims to investigate the impact of aggregation pattern of two Aβ peptides on the structural stability of the Aβ42 dimer and its disruption to the biological membrane. The MD results demonstrate that the NCCN, CNNC and NCNC have the larger structural fluctuation at the N-terminus of Aβ42 peptide, where the β-strand structure converts into the coil structure. The loss of the N-terminal β-strand further impairs the aggregate ability of Aβ42 dimer. In addition, inserting Aβ42 dimer into the membrane can considerably decrease the average APL of DPPC membrane. Moreover this decrease effect is largely dependent on the distance to the location of Aβ42 dimer and its secondary structure forms. Based on the results, the 2NCb is considered as a stable dimeric unit for aggregating the larger Aβ42 oligomer, and has a potent ability to disrupt the membrane.

Structure modification of particle track membranes

International Nuclear Information System (INIS)

Lueck, H.B.; Gemende, B.; Heinrich, B.

1991-01-01

Three different structure modifications were studied in order to improve the flux and dirt loading capacity of particle track membranes without affecting their retention characteristic. Divergent irradiation is a very effective tool for decreasing the number of multiple pores and increasing the porosity up to 20 per cent. The technique leads to a remarkable but not efficient enhancement of the surface porosity. Improved surface porosity produced by a double irradiation technique turns out to be very effective with respect to the filtration performance. (author)

Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg2+

International Nuclear Information System (INIS)

Du, Z.; Boyer, P.D.

1989-01-01

The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg 2+ with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg 2+ is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg 2+ released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg 2+ the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg 2+ and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg 2+ . The replacement of [ 3 H]ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg 2+ and ADP bound at one catalytic site without P i block catalysis by all three enzyme sites. The inhibited form induced by Mg 2+ and ADP may occur only under laboratory conditions and not have an in vivo role

Axionic membranes

International Nuclear Information System (INIS)

Aurilia, A.; Spallucci, E.

1992-01-01

A metal ring removed from a soap-water solution encloses a film of soap which can be mathematically described as a minimal surface having the ring as its only boundary. This is known to everybody. In this letter we suggest a relativistic extension of the above fluidodynamic system where the soap film is replaced by a Kalb-Ramand gauge potential B μν (x) and the ring by a closed string. The interaction between the B μν field and the string current excites a new configuration of the system consisting of a relativistic membrane bounded by the string. We call such a classical solution of the equation of motion an axionic membrane. As a dynamical system, the axionic membrane admits a Hamilton-Jacobi formulation which is an extension of the HJ theory of electromagnetic strings. (orig.)

Communication Between the Cell Membrane and the Nucleus: Role of Protein Compartmentalization

Energy Technology Data Exchange (ETDEWEB)

Lelievre, Sophie A; Bissell, Mina J

1998-10-21

Understanding how the information is conveyed from outside to inside the cell is a critical challenge for all biologists involved in signal transduction. The flow of information initiated by cell-cell and cell-extracellular matrix contacts is mediated by the formation of adhesion complexes involving multiple proteins. Inside adhesion complexes, connective membrane skeleton (CMS) proteins are signal transducers that bind to adhesion molecules, organize the cytoskeleton, and initiate biochemical cascades. Adhesion complex-mediated signal transduction ultimately directs the formation of supramolecular structures in the cell nucleus, as illustrated by the establishment of multi complexes of DNA-bound transcription factors, and the redistribution of nuclear structural proteins to form nuclear subdomains. Recently, several CMS proteins have been observed to travel to the cell nucleus, suggesting a distinctive role for these proteins in signal transduction. This review focuses on the nuclear translocation of structural signal transducers of the membrane skeleton and also extends our analysis to possible translocation of resident nuclear proteins to the membrane skeleton. This leads us to envision the communication between spatially distant cellular compartments (i.e., membrane skeleton and cell nucleus) as a bidirectional flow of information (a dynamic reciprocity) based on subtle multilevel structural and biochemical equilibria. At one level, it is mediated by the interaction between structural signal transducers and their binding partners, at another level it may be mediated by the balance and integration of signal transducers in different cellular compartments.

Direct quantification of negatively charged functional groups on membrane surfaces

KAUST Repository

Tiraferri, Alberto; Elimelech, Menachem

2012-01-01

groups at the surface of dense polymeric membranes. Both techniques consist of associating the membrane surface moieties with chemical probes, followed by quantification of the bound probes. Uranyl acetate and toluidine blue O dye, which interact

Structure of alanine racemase from Oenococcus oeni with bound pyridoxal 5′-phosphate

International Nuclear Information System (INIS)

Palani, Kandavelu; Burley, Stephen K.; Swaminathan, Subramanyam

2012-01-01

Alanine racemase from O. oeni exists as a dimer in the crystal structure. Both monomers contribute to the two active sites present, one for each monomer. The crystal structure of alanine racemase from Oenococcus oeni has been determined at 1.7 Å resolution using the single-wavelength anomalous dispersion (SAD) method and selenium-labelled protein. The protein exists as a symmetric dimer in the crystal, with both protomers contributing to the two active sites. Pyridoxal 5′-phosphate, a cofactor, is bound to each monomer and forms a Schiff base with Lys39. Structural comparison of alanine racemase from O. oeni (Alr) with homologous family members revealed similar domain organization and cofactor binding

The molecular architecture of the chloroplast thylakoid membrane

Energy Technology Data Exchange (ETDEWEB)

Stefansson, H.

1996-08-01

Non-detergent procedure for isolation of sub-thylakoid vesicle populations derived from different structural domains of the chloroplast thylakoid membrane has been developed. Sub-thylakoid vesicles representing the grana, grana core, stroma lamellae, and the grana margins have been isolated and their protein composition has been investigated. Furthermore a novel non-detergent procedure for investigating the pigment composition of photosynthetic complexes located in the different structural domains has been developed. This procedure circumvents selective extractions, an perturbing effect often combined with detergent isolations of membrane bound protein complexes. The fractionation experiments show that the NADPH dehydrogenase, suggested to operate as NADPH or ferredoxin-plastoquinone oxidoreductase in cyclic electron transport around photosystem I, is stoichiometrically depleted on photosystem I basis in the grana domain. The fractionation studies are consistent with the model of the thylakoid membrane where the photosystems in the grana are operating in a linear electron transport whereas the site of cyclic electron transport is in the stroma lamellae. It is suggested that partial destacking of grana, as a result of light-induced protein phosphorylation, may promote the exposure of the granal photosystem I centers to the chloroplast stroma and thereby enhance their participation in cyclic electron transport activity. 146 refs, 18 figs

NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1.

Science.gov (United States)

Güttler, Thomas; Madl, Tobias; Neumann, Piotr; Deichsel, Danilo; Corsini, Lorenzo; Monecke, Thomas; Ficner, Ralf; Sattler, Michael; Görlich, Dirk

2010-11-01

Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP-CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an α-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.

Cell Secretion: Current Structural and Biochemical Insights

Directory of Open Access Journals (Sweden)

Saurabh Trikha

2010-01-01

Full Text Available Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

International Nuclear Information System (INIS)

Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Urzhumtsev, Alexandre; Gualerzi, Claudio O.; Klaholz, Bruno P.

2013-01-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

Energy Technology Data Exchange (ETDEWEB)

Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

2013-06-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

The asymmetrical structure of Golgi apparatus membranes revealed by in situ atomic force microscope.

Directory of Open Access Journals (Sweden)

Haijiao Xu

Full Text Available The Golgi apparatus has attracted intense attentions due to its fascinating morphology and vital role as the pivot of cellular secretory pathway since its discovery. However, its complex structure at the molecular level remains elusive due to limited approaches. In this study, the structure of Golgi apparatus, including the Golgi stack, cisternal structure, relevant tubules and vesicles, were directly visualized by high-resolution atomic force microscope. We imaged both sides of Golgi apparatus membranes and revealed that the outer leaflet of Golgi membranes is relatively smooth while the inner membrane leaflet is rough and covered by dense proteins. With the treatment of methyl-β-cyclodextrin and Triton X-100, we confirmed the existence of lipid rafts in Golgi apparatus membrane, which are mostly in the size of 20 nm -200 nm and appear irregular in shape. Our results may be of significance to reveal the structure-function relationship of the Golgi complex and pave the way for visualizing the endomembrane system in mammalian cells at the molecular level.

The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health.

Science.gov (United States)

Ibarguren, Maitane; López, David J; Escribá, Pablo V

2014-06-01

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Polymer-metal organic frameworks (MOFs) mixed matrix membranes for gas separation applications

NARCIS (Netherlands)

Shahid, S.

2015-01-01

The performance of polymeric membranes is often limited by a trade-off between membrane permeability and selectivity, the so-called Robeson upper bound. Additionally, in high pressure CO2 capture applications, excessive swelling of the polymer membrane often leads to plasticization resulting in

Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

Science.gov (United States)

Wang, Jimin; Li, Yue; Modis, Yorgo

2014-01-01

The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

E.s.r. radiation studies of erythrocyte membrane-haemoglobin interaction

International Nuclear Information System (INIS)

Koter, M.; Kowalska, M.A.; Leyko, W.; Waterman, M.

1977-01-01

The dependence of the yield of free radicals in gamma-irradiated, freeze-dried erythrocyte membranes on their haemoglobin content was studied. A non-monotonous relationship was found, different from that observed in mixtures of freeze-dried membranes and haemoglobin, which suggests the existence of radiation-energy transfer between the membranes and bound haemoglobin. (author)

Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg sup 2+

Energy Technology Data Exchange (ETDEWEB)

Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

1989-01-24

The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg{sup 2+} with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg{sup 2+} is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg{sup 2+} released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg{sup 2+} the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg{sup 2+} and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg{sup 2+}. The replacement of ({sup 3}H)ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg{sup 2+} and ADP bound at one catalytic site without P{sub i} block catalysis by all three enzyme sites. The inhibited form induced by Mg{sup 2+} and ADP may occur only under laboratory conditions and not have an in vivo role.

Fine-Tuned Intrinsically Ultramicroporous Polymers Redefine the Permeability/Selectivity Upper Bounds of Membrane-Based Air and Hydrogen Separations

KAUST Repository

Swaidan, Raja

2015-08-20

Intrinsically ultramicroporous (<7 Å) polymers represent a new paradigm in materials development for membrane-based gas separation. In particular, they demonstrate that uniting intrachain “rigidity”, the traditional design metric of highly permeable polymers of intrinsic microporosity (PIMs), with gas-sieving ultramicroporosity yields high-performance gas separation membranes. Highly ultramicroporous PIMs have redefined the state-of-the-art in large-scale air (e.g., O2/N2) and hydrogen recovery (e.g., H2/N2, H2/CH4) applications with unprecedented molecular sieving gas transport properties. Accordingly, presented herein are new 2015 permeability/selectivity “upper bounds” for large-scale commercial membrane-based air and hydrogen applications that accommodate the substantial performance enhancements of recent PIMs over preceding polymers. A subtle balance between intrachain rigidity and interchain spacing has been achieved in the amorphous microstructures of PIMs, fine-tuned using unique bridged-bicyclic building blocks (i.e., triptycene, ethanoanthracene and Tröger’s base) in both ladder and semiladder (e.g., polyimide) structures.

Upper-Lower Bounds Candidate Sets Searching Algorithm for Bayesian Network Structure Learning

Directory of Open Access Journals (Sweden)

Guangyi Liu

2014-01-01

Full Text Available Bayesian network is an important theoretical model in artificial intelligence field and also a powerful tool for processing uncertainty issues. Considering the slow convergence speed of current Bayesian network structure learning algorithms, a fast hybrid learning method is proposed in this paper. We start with further analysis of information provided by low-order conditional independence testing, and then two methods are given for constructing graph model of network, which is theoretically proved to be upper and lower bounds of the structure space of target network, so that candidate sets are given as a result; after that a search and scoring algorithm is operated based on the candidate sets to find the final structure of the network. Simulation results show that the algorithm proposed in this paper is more efficient than similar algorithms with the same learning precision.

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

Crosslinked copolyazoles with a zwitterionic structure for organic solvent resistant membranes

KAUST Repository

Chisca, Stefan

2015-01-01

The preparation of crosslinked membranes with a zwitterionic structure based on a facile reaction between a newly synthesized copolyazole with free OH groups and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) is reported. The new OH-functionalized copolyazole is soluble in common organic solvents, such as tetrahydrofuran (THF), dimethylsulfoxide (DMSO), N,N′-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP) and can be easily processed by phase inversion. After crosslinking with GPTMS, the membranes acquire high solvent resistance. We show the membrane performance and the influence of the crosslinking reaction conditions on the thermal stability, surface polarity, pore morphology, and solvent resistance. By using UV-spectroscopy we monitored the solvent resistance of the membranes in four aggressive solvents (THF, DMSO, DMF and NMP) for 30 days. After this time, only minor changes (less than 2%) were detected for membranes subjected to a crosslinking reaction for 6 hours or longer. Our data suggest that the novel crosslinked membranes can be used for industrial applications in wide harsh environments in the presence of organic solvents.

Different Structures of PVA Nano fibrous Membrane for Sound Absorption Application

International Nuclear Information System (INIS)

Mohrova, J.; Kalinova, K.

2012-01-01

The thin nano fibrous layer has different properties in the field of sound absorption in comparison with porous fibrous material which works on a principle of friction of air particles in contact with walls of pores. In case of the thin nano fibrous layer, which represents a sound absorber here, the energy of sonic waves is absorbed by the principle of membrane resonance. The structure of the membrane can play an important role in the process of converting the sonic energy to a different energy type. The vibration system acts differently depending on the presence of smooth fibers in the structure, amount of partly merged fibers, or structure of polymer foil as extreme. Polyvinyl alcohol (PVA) was used as a polymer because of its good water solubility. It is possible to influence the structure of nano fibrous layer during the production process thanks to this property of polyvinyl alcohol.

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

International Nuclear Information System (INIS)

Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi; Ohno-Iwashita, Yoshiko

2006-01-01

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with β-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

Science.gov (United States)

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function

Directory of Open Access Journals (Sweden)

Jakob Andersson

2016-05-01

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

Inhibition of neutrophil migration by aggregated immunoglobulin attached to micropore membranes.

Science.gov (United States)

Kemp, A S; Brown, S

1980-01-01

The effect of substrate-bound immunoglobulin on neutrophil migration was examined. Immunoglobulin aggregates bound to micropore membranes inhibited the neutrophil response to a chemotactic stimulus. This inhibition was reversed by the presence of aggregates in suspension suggesting competition between substrate-bound and free aggregates for neutrophil surface binding sites. The immobilization of neutrophils by substrate-bound aggregated immunoglobulin suggests a mechanism for the accumulation of neutrophils at sites of immune complex deposition and tissue-bound antibodies in vivo. PMID:7380477

Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes.

Science.gov (United States)

Brand, S H; Holtzman, E J; Scher, D A; Ausiello, D A; Stow, J L

1996-05-01

Heterotrimeric G protein alpha-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the G alpha i-3 subunit to Golgi membranes. G alpha i-3 was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha i-3 was not myristoylated. In contrast to endogenous G alpha 1-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha i-3 did not attach to membranes; it was localized by immunofluorescence in the cytoplasm of LLC-PK1 cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-alpha i-3 to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha i-3 in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha i-3. These data are consistent with a model in which G alpha i-3 utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.; Choi, Seung Hak

2012-01-01

. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane

Structure determination of an integral membrane protein at room temperature from crystals in situ

International Nuclear Information System (INIS)

Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

2015-01-01

The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines

Structure determination of an integral membrane protein at room temperature from crystals in situ

Energy Technology Data Exchange (ETDEWEB)

Axford, Danny [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Foadi, James [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Hu, Nien-Jen; Choudhury, Hassanul Ghani [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Iwata, So [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Kyoto University, Kyoto 606-8501 (Japan); Beis, Konstantinos [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Alguel, Yilmaz, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom)

2015-05-14

The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

Taurocholate transport by brush-border membrane vesicles from the developing rabbit ileum: Structure/function relationships

International Nuclear Information System (INIS)

Schwarz, S.M.; Watkins, J.B.; Ling, S.C.

1990-01-01

To examine the ontogenesis of bile acid transport in the rabbit ileum, brush-border membrane vesicles (12- to 20-fold purified) were prepared from 14- to 49-day-old animals. Taurocholate uptake was characterized by the emergence of secondary active, Na(+)-dependent transport at the start of weaning (21 days). Transient intravesicular accumulation (overshoot) of taurocholate occurred at 5-10 s of incubation, and the overshoot maximum increased significantly from 21 days (349.2 +/- 22.4 nmol/mg protein) to 35 days (569.0 +/- 84.3 nmol/mg protein; p less than 0.001), without further increase at maturity (49 days, not equal to 607.6 +/- 136.7 nmol/mg protein). No significant taurocholate active uptake component was noted at 14 days; however, ileal vesicles from sucklings showed carrier-mediated, Na+ D-glucose cotransport. In greater than or equal to 35-day-old rabbits, osmolarity studies at 20 s of incubation showed that only approximately 12% of [14C]taurocholate uptake was secondary to bile acid-to-membrane binding. Conversely, at 20 min, greater than 95% of radiolabel incorporation represented solute bound to the external and/or internal membrane surface. Arrhenius plots establish brush-border membrane taurocholate uptake as an intrinsic, lipid-dependent process, with a slope discontinuity between 24 and 28 degrees C, similar to the membrane lipid thermotropic transition region. Steady-state fluorescence polarization studies (1,6-diphenyl-1,3,5-hexatriene) demonstrate a temporal association between the maturation of taurocholate uptake and age-related decreases in ileal brush-border membrane fluidity. These data indicate that maturation of bile acid secondary active transport in the rabbit ileum may be regulated, at least in part, by changes in brush-border membrane lipid dynamics

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

Science.gov (United States)

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

Structural characterization of Bacillus subtilis membrane protein Bmr: an in silico approach.

Science.gov (United States)

Nargotra, Amit; Rukmankesh; Ali, Shakir; Koul, Surrinder

2014-01-01

Efflux pump--a membrane protein belonging to Major Facilitator (MF) family and associated with Multi Drug Resistance (MDR) has been a major factor in drug resistance of bacteria. In the era when no new effective antibiotic had been reported for years, the detailed study of these membrane proteins became imperative in order to improve the efficacy of existing drugs. The Bacillus subtilis membrane protein Bmr belongs to the super family of major facilitator proteins and is one of the first-discovered bacterial multidrug-efflux transporters. Development of Bmr inhibitors (B. subtilis) for least resistance, better drug sustainability and effective cellular activity requires three dimensional structure of this protein which has not yet been determined. In this communication structural characterization of this important efflux pump has been attempted using in silico approaches. The modeled structure of Bmr has been found to have 12 main helical segments interspersed by loops of variable lengths at regular intervals with both N- and C-termini on the same side of membrane. Docking of the known inhibitor reserpine on to the predicted structure of Bmr and its mutants signified the importance of the residues Phe143, Val286 and Phe306 in the interaction with the ligand. Besides this, the role of Arg313 and Phe309 in the H-bond formation and π-π interaction respectively, with reserpine was the new significant finding based on the interaction studies. The structure elucidation of Bmr and the role of these residues in binding to the ligand are expected to have a great impact on the efflux pump inhibition studies around the world and hence in the efficiency of the existing antibiotic drugs.

Fabrication of functional structures on thin silicon nitride membranes

NARCIS (Netherlands)

Ekkels, P.; Tjerkstra, R.W.; Krijnen, Gijsbertus J.M.; Berenschot, Johan W.; Brugger, J.P.; Elwenspoek, Michael Curt

A process to fabricate functional polysilicon structures above large (4×4 mm2) thin (200 nm), very flat LPCVD silicon rich nitride membranes was developed. Key features of this fabrication process are the use of low-stress LPCVD silicon nitride, sacrificial layer etching, and minimization of

Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

International Nuclear Information System (INIS)

Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

2008-01-01

Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 (angstrom) resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

International Nuclear Information System (INIS)

Bienz, K.; Egger, D.; Troxler, M.; Pasamontes, L.

1990-01-01

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed

Cell-secreted flavins bound to membrane cytochromes dictate electron transfer reactions to surfaces with diverse charge and pH.

Science.gov (United States)

Okamoto, Akihiro; Kalathil, Shafeer; Deng, Xiao; Hashimoto, Kazuhito; Nakamura, Ryuhei; Nealson, Kenneth H

2014-07-11

The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.

The membrane fraction of homogenized rat kidney contains an enzyme that releases epidermal growth factor from the kidney membranes

DEFF Research Database (Denmark)

Nexø, Ebba; Poulsen, Steen Seier

1991-01-01

shows that the membrane fraction of homogenized rat kidney contains an enzyme that releases immuno and receptor reactive EGF from the kidney membranes when incubated at 37 degrees C. Gel filtration shows that the EGF reactivity released from the membranes is similar to the EGF reactivity in rat urine......High levels of epidermal growth factor (EGF) are excreted in the urine and high levels of mRNA for the EGF-precursor have been demonstrated in the kidney. The EGF-precursor is a membrane bound peptide in the kidney, but little is known about the renal processing of the precursor. The present study...

Bounded elements in Locally C*-algebras

International Nuclear Information System (INIS)

El Harti, Rachid

2001-09-01

In order to get more useful information about Locally C*-algebras, we introduce in this paper the notion of bounded elements. First, we study the connection between bounded elements and spectrally bounded elements. Some structural results of Locally C*-algebras are established in Theorems 1 , 2 and 3. As an immediate consequence of Theorem 3, we give a characterization of the connected component of the identity in the group of unitary elements for a Locally C*-algebra. (author)

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

Science.gov (United States)

Isabet, Tatiana; Montagnac, Guillaume; Regazzoni, Karine; Raynal, Bertrand; El Khadali, Fatima; England, Patrick; Franco, Michel; Chavrier, Philippe; Houdusse, Anne; Ménétrey, Julie

2009-09-16

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

Efficient DNP NMR of Membrane Proteins: Sample Preparation Protocols, Sensitivity, and Radical Location

Science.gov (United States)

Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V.; Hong, Mei

2016-01-01

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

Energy Technology Data Exchange (ETDEWEB)

Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo [Massachusetts Institute of Technology, Department of Chemistry (United States); Sergeyev, Ivan V. [Bruker Biospin (United States); Hong, Mei, E-mail: meihong@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

2016-03-15

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

Science.gov (United States)

Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.

Science.gov (United States)

Schaffer, Miroslava; Mahamid, Julia; Engel, Benjamin D; Laugks, Tim; Baumeister, Wolfgang; Plitzko, Jürgen M

2017-02-01

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes.

Science.gov (United States)

Szekely, Or; Steiner, Ariel; Szekely, Pablo; Amit, Einav; Asor, Roi; Tamburu, Carmen; Raviv, Uri

2011-06-21

In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of ∼10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar

Asymmetric block copolymer membranes with ultrahigh porosity and hierarchical pore structure by plain solvent evaporation

KAUST Repository

Yu, H.

2016-09-14

Membranes with a hierarchical porous structure could be manufactured from a block copolymer blend by pure solvent evaporation. Uniform pores in a 30 nm thin skin layer supported by a macroporous structure were formed. This new process is attractive for membrane production because of its simplicity and the lack of liquid waste.

Asymmetric block copolymer membranes with ultrahigh porosity and hierarchical pore structure by plain solvent evaporation

KAUST Repository

Yu, H.; Qiu, Xiaoyan; Behzad, Ali Reza; Musteata, Valentina-Elena; Smilgies, D.-M.; Nunes, Suzana Pereira; Peinemann, Klaus-Viktor

2016-01-01

Membranes with a hierarchical porous structure could be manufactured from a block copolymer blend by pure solvent evaporation. Uniform pores in a 30 nm thin skin layer supported by a macroporous structure were formed. This new process is attractive for membrane production because of its simplicity and the lack of liquid waste.

Development of Hydrogen Separation Module with Structured Catalyst for Use in Membrane Reformer

International Nuclear Information System (INIS)

Isamu Yasuda; Tatsuya Tsuneki; Yoshinori Shirasaki; Toru Shimamori; Hidekazu Shigaki; Hiroyuki Tanaka

2006-01-01

A new type of hydrogen separation module for use in a membrane reformer was proposed and developed. The new module, what we call MOC (Membrane On Catalyst), was designed to have a membrane of palladium-based alloy prepared on the surface of the tubular structured catalyst that has catalytic activity for steam reforming reaction, thermal expansion matching with the membrane material, proper porosity, mechanical strength and thermal conductivity. The best composition of the structured catalyst was identified in the composites of metallic Ni and YSZ (Yttria-Stabilized Zirconia). A hydrogen separation module was manufactured by electroless plating of Pd with thickness of 7 to 15 microns on the surface of porous sintered tube of Ni-YSZ with an approximate size of 9 mm in diameter and 100 mm in length. The hydrogen permeability measurements have shown hydrogen flux of 25 to 35 cc/min at 550 to 600 C, which is higher than the permeability of the conventional modules using rolled Pd film. (authors)

Probing protein-lipid interactions by FRET between membrane fluorophores

Science.gov (United States)

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.

Physico-mechanical and structural properties of eggshell membrane gelatin- chitosan blend edible films

DEFF Research Database (Denmark)

Mohammadi, Reza; Mohammadifar, Mohammad Amin; Rouhi, Milad

2018-01-01

This study investigated the physico-mechanical and structural properties of composite edible films based on eggshell membrane gelatin (G) and chitosan (Ch) (75G:25Ch, 50G:50Ch, 25G:75Ch). The results demonstrated that the addition of Ch increased elongation at break significantly (p< 0.05), but r......This study investigated the physico-mechanical and structural properties of composite edible films based on eggshell membrane gelatin (G) and chitosan (Ch) (75G:25Ch, 50G:50Ch, 25G:75Ch). The results demonstrated that the addition of Ch increased elongation at break significantly (p... interactions introduced by the addition of chitosan to eggshell membrane gelatin as new resources could improve the films’ functional properties....